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(Hypertension. 1995;26:725.)
© 1995 American Heart Association, Inc.

Articles

G Protein Alterations in Hypertension and Aging

R.D. Feldman; C.M. Tan; J. Chorazyczewski

From the Departments of Medicine and Pharmacology and Toxicology, University of Western Ontario, London, Ontario, Canada.

Correspondence to Dr R.D. Feldman, 6-L13 University Hospital, PO Box 5339, 339 Windermere Rd, London, Ontario, Canada N6A 5A5.

	Abstract

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Abstract Defective vasodilator function could be important in the pathogenesis and/or maintenance of the hypertensive state and the predisposition of the elderly to hypertension. Impaired ß-adrenergic–mediated vasodilation and reduced lymphocyte ß-adrenergic activation of adenyl cyclase have been demonstrated both in aging and with hypertension. The cellular mechanisms responsible for these alterations remain unclear. To determine if these defects may be due to alterations in guanine nucleotide regulatory proteins (G proteins) that link receptor activation with effector function, we assessed (1) human lymphocyte adenyl cyclase activity, (2) stimulatory G proteins by cholera toxin–mediated [³²P]ADP ribosylation and, in hypertensive subjects, with _s-specific and ß-subunit antisera, and (3) inhibitory G proteins by pertussis toxin–mediated [³²P]ADP ribosylation and, in older subjects, with _i1,2- and ß-subunit–specific antisera. Lymphocytes from older subjects and from hypertensive subjects demonstrated a comparable reduction in isoproterenol-stimulated adenyl cyclase. However, aluminum fluoride–stimulated activity was reduced only in lymphocytes from hypertensive subjects. Furthermore, aluminum fluoride–stimulated activity was inversely correlated with mean arterial pressure. In lymphocytes from younger hypertensive subjects, cholera toxin–mediated labeling was significantly reduced; however, stimulatory G protein labeling by immunodetection was unaltered. In lymphocytes from older subjects, cholera toxin–mediated labeling was not altered; however, pertussis toxin–mediated labeling was significantly increased. In contrast, inhibitory G protein labeling by immunodetection was unaltered. Overall, the study suggests alterations of G protein function in hypertension and aging. In both conditions, stimulation of adenyl cyclase is impaired. However, these defects are associated with divergent alterations in stimulatory and inhibitory G proteins.

Key Words: G proteins • adenyl cyclase • hypertension

	Introduction

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Functional alterations in vascular signal transduction mechanisms may be important in the pathogenesis and maintenance of elevated vascular resistance in hypertension¹ and in the increased incidence of hypertension with aging.^{2 3} We and others have focused on functional impairment of receptor systems linked to vasodilation as a potential mechanism underlying the elevation in vascular resistance in hypertension and aging.

Impaired ß-adrenergic–mediated vasodilation has been reported both with aging^{4 5 6 7} and in hypertensive subjects.^{1 8} Furthermore, in both conditions, the defect has been shown to be functional (at least in part) and reversible.^{1 7 9}

It has been suggested that the impairment in ß-adrenergic–mediated vasodilation is due to reduced ß-adrenergic–mediated adenyl cyclase activation (see below). Therefore, attention has been focused on the transmembrane signal transduction system linked to activation of adenyl cyclase. This consists of the catalytic subunit, the receptor, and guanine nucleotide regulatory proteins (G proteins) (reviewed in References 10 and 11^{10 11} ). The stimulatory G protein (G_s) is directly linked to adenyl cyclase activation. An inhibitory G protein (G_i) mediates the effects of receptors acting through inhibition of adenyl cyclase. Additionally, G_i may exert a tonic inhibitory effect on the stimulation of adenyl cyclase.^{12 13 14 15}

In hypertension and aging, the lymphocyte has been used as a model for the human vascular ß-adrenoceptor–G protein adenyl cyclase complex. The usefulness of this model is based in part on the observation that in aging and hypertension, both vascular and lymphocyte ß-adrenergic responsiveness are impaired (as reported by most but not all investigators; reviewed in References 16 through 18^{16 17 18} ).* Furthermore, vascular and lymphocyte ß-adrenergic responsiveness are regulated in parallel, at least under some conditions, in older and in hypertensive subjects (ie, by dietary salt restriction^{1 7 8 9} ). Specifically, we have found in several studies of younger, white, borderline or mildly hypertensive subjects receiving a dietary salt intake >150 mg/d^{9 20} that lymphocyte receptor–mediated adenyl cyclase activation is impaired. Furthermore, we and others have demonstrated in this subgroup of hypertensive subjects fed a normal to high dietary salt intake that impairment in adenyl cyclase activation is associated with reduced ß-adrenoceptor affinity for agonists, suggesting an alteration in receptor–G protein interactions.^{8 20} A similar defect has been reported in older, normotensive subjects.^{21 22}

On the basis of these findings, we examined G protein properties in lymphocytes from younger hypertensive subjects, older normotensive subjects, and younger normotensive control subjects. Data to be presented demonstrated alterations in G proteins both in hypertensive and in older subjects. However, the pattern of alterations in each group is distinct.

	Methods

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Subject Protocol
Younger and older normotensive subjects and younger borderline or mildly hypertensive subjects were studied. Younger normotensive subjects were between the ages of 20 to 36 years, healthy, and not taking any medications on a regular basis or any medications for at least 1 month before study. Older normotensive subjects were between the ages of 45 and 63 years (comparable to the age of older subjects in whom we have previously reported alterations in both vascular and lymphocyte ß-adrenergic responsiveness⁷ ), healthy, and not taking any medications on a regular basis or any medications for at least 1 month before study. Hypertensive subjects were white, between the ages of 22 and 36 years, otherwise healthy, and had neither renal nor cardiovascular complications. Hypertensive subjects had not taken any antihypertensive medications (or any other medications) for at least 1 month before study. The criteria for classifying blood pressure status of subjects were as described previously.¹ The borderline or mildly hypertensive subjects had pressures >140/90 mm Hg on at least 20% of daytime automatic ambulatory blood pressure readings (Spacelab model 90207, the Table).

View this table: [in this window] [in a new window]   Table 1. Alterations in Age, Blood Pressure, and Urinary Sodium Excretion in Study Groups

All subjects were instructed to maintain a high sodium intake for 3 days before study. Compliance with dietary instruction was assessed by urinary sodium determinations based on overnight collections (the Table). For toxin-mediated labeling assays, 13 older subjects and 7 hypertensive subjects were studied on a paired basis, ie, on each study day, an older or hypertensive subject was studied concurrently with a younger normotensive control subject. In a second protocol, blood samples were obtained under identical conditions as above from 8 of the older subjects and 5 of the younger normotensive subjects for immunochemical assessment of G_i proteins and from 6 of the hypertensive subjects and 7 of the younger normotensive subjects for immunochemical assessment of alterations in G_s proteins. Last, in a third protocol, blood samples were obtained from an additional group of 9 younger normotensive subjects, 7 older normotensive subjects, and 7 younger hypertensive subjects for assessment of lymphocyte adenyl cyclase activity. All protocols were approved by the Human Subjects Review Committee, University of Western Ontario. Informed consent was obtained from each subject before study.

G proteins were assessed in mononuclear leukocyte preparations. Mononuclear leukocytes were separated from whole blood by the method of Boyum²³ and as described previously.⁷ For ADP ribosylation studies, samples were resuspended in a solution of (mmol/L) Tris-HCl 5, EDTA 3, NaCl 155; pH 7.6 at room temperature. For immunoblotting, cell pellets were frozen at -70°C before assay. For adenyl cyclase assays, samples were resuspended in Hanks’ balanced salt solution (HBSS without Mg²⁺ or Ca²⁺), centrifuged for 10 minutes at 400g, and resuspended in HBSS. Cells were made permeable with digitonin (2 µg/mL) as previously described,⁷ washed as above, and resuspended at a concentration of 20x10⁶ cells/mL in HBSS for assays of adenyl cyclase activity.

Assessment of Toxin-Mediated G Protein Labeling
The ADP ribosylation of G proteins by pertussis toxin (PT) was carried out according to the method of Kopf and Woolkalis.²⁴ PT (10 µg/mL) was preactivated in a solution of 50 mmol/L HEPES, pH 8.0, 1 mg/mL bovine serum albumin (BSA), 0.125 g/100 mL SDS, 20 mmol/L DL-dithiothreitol at 30°C for 30 minutes. Preactivated PT and [³²P]NAD (50 to 100 µCi/mL) with NAD (50 µmol/L) in a volume of 20 µL were added to an assay mixture of 80 µL containing the cell suspension with 1 mmol/L EDTA and 10 mmol/L thymidine. Addition of the "preactivation mixtures" (with SDS) to the cell suspension resulted in cell lysis. The samples were incubated at 30°C for 30 minutes, and the reaction was terminated by adding 1 mL of an ice-cold solution of (mmol/L)Tris-HCl 5, EDTA 3; pH 7.6. The lysates were recovered by centrifugation for 15 minutes at 12 000g. The pellet was washed with the same buffer and was centrifuged again. SDS-PAGE sample buffer (described below) was added, and each sample was boiled for 5 minutes.

The ADP ribosylation of G proteins by cholera toxin (CT) was carried out according to the method of Gill and Woolkalis.²⁵ CT (100 µg/mL) was activated in a solution of 50 mmol/L HEPES, pH 8.0, 1 mg/mL BSA, 0.125% SDS and 130 mmol/L NaCl at 30°C for 30 minutes. Preactivated CT and [³²P]NAD (50 to 100 µCi/mL) with NAD (50 µmol/L) were added in a volume of 20 µL to the cell suspension in a solution with 1 mmol/L EDTA, 100 mmol/L NaCl, 100 µmol/L GTP and 10 mmol/L thymidine to a total volume of 100 µL. The reaction was stopped and lysates were collected as described above.

SDS-PAGE was performed using the procedure described by Laemmli.²⁶ Briefly, lysates (25 to 50 µg of protein; protein content was matched in each pair of samples analyzed) were dissolved in 50 µL of sample buffer containing 125 mmol/L Tris-HCl, pH 6.8, 20% glycerol, 40 g/L SDS, 100 g/L 2-mercaptoethanol, and 0.25 g/L bromphenol blue and boiled for 5 minutes before application to the gel. Protein molecular weight markers were dissolved in the same buffer. A 12% polyacrylamide running gel with a 4% stacking gel was used for all studies (model SE-400 gel apparatus, Hoefer). Electrophoresis was performed at a fixed current of 10 mA per gel slab for 15 to 18 hours. Gels were stained on 2 g/L Coomassie brilliant blue R-250, 50% methanol, 10% acetic acid for 30 minutes followed by rapid destaining in 50% methanol, 10% acetic acid for 3 to 4 hours. The gels were then air-dried overnight and exposed to x-ray film for 2 to 5 days at -80°C (Kodak XAR). G protein labeling was quantified by laser densitometric assessment of toxin-specific labeling (LKB 2222-020, Pharmacia-LKB Biotechnology) in films demonstrating submaximal exposure of toxin-specific bands in both control and experimental samples.

Initial studies indicated that these conditions resulted in maximal toxin-mediated labeling, ie, that pretreatment of lysates with up to five times higher toxin concentrations and for up to twofold longer durations of incubation did not result in significantly greater labeling. Furthermore, under these conditions, density of exposure was linearly correlated with amount of protein loaded.

Assessment of G Proteins by Immunoblotting
Immunochemical labeling of - and ß-subunits of mononuclear leukocyte G proteins was performed by Western blotting. Briefly, aliquots of the frozen (-70°C) human mononuclear leukocyte pellets (containing 20 µg of protein) were thawed by addition of sample buffer (as above) and boiled for 5 minutes before application to gel. Protein content was matched for all samples loaded (to approximately 12 µg). After electrophoresis (1.5 hours at 140 V), the gels were soaked for 20 minutes in transfer buffer (48 mmol/L Tris, 39 mmol/L glycine, 0.375 g/L SDS, 20% methanol). Proteins were transferred from the gels to presoaked nitrocellulose membranes (Immobilon-P transfer membranes, Millipore) at 100 V for 45 minutes. Membranes were blocked overnight in a solution of 10 g/L BSA in transfer buffer at pH 7.4 with 0.1% Tween 20 and 500 mmol/L NaCl. Immunoblotting was carried out by incubating the membranes (1 hour at room temperature) with _s-, _o-, _1,2-, _i,3-, or ß-subunit–specific antisera (RM/1, SW/1, AS/7, EC/2, and GC/2, respectively; all obtained from DuPont–New England Nuclear Corp) in a dilution of l:5000 in "blocking buffer." Membranes were washed once and then incubated for 20 minutes with peroxidase-conjugated goat anti-rabbit IgG at a 1:20 000 dilution. Immunoreactivity was detected with the enhanced chemiluminescence detection system after exposure to x-ray film (Kodak XLS). Intensity of the specific band was assessed densitometrically (as above). On each study day, 13 samples were assessed on two membranes, processed simultaneously. Differences in transfer efficiency between the two simultaneously processed membranes were normalized by the extent of labeling of a bovine brain G protein standard run with each gel/membrane.

G protein labeling as assessed from Western blots of lymphocytes from hypertensive subjects and from elderly subjects was expressed relative to the average extent of labeling in lymphocytes from the younger normotensive subjects studied concurrently.

For each of the G protein comparisons performed, (ie, comparisons of _s-, -, and ß-subunit labeling), the panel of 13 samples was assessed on two to four separate occasions. In this way, a standardized coefficient of variation was determined. On repeated measurements, the average coefficients of variation (for an individual sample) were 12% for _i-, 21% for _s-, and 7% for ß-subunit labeling.

Assays of Adenyl Cyclase Activity in Permeable Lymphocytes
Assays of adenyl cyclase activity were performed on permeable cell preparations according to our previously published methods.⁷ Permeable cells resuspended in buffer A were added in an aliquot of 40 µL to give a final incubation volume of 100 µL with 1 µCi [³²P] ATP, 0.3 mmol/L ATP, 2 mmol/L MgSO₄, 0.1 mmol/L cAMP (used in lieu of a phosphodiesterase inhibitor), 5 mmol phosphoenolpyruvate, 40 µg/mL pyruvate kinase, and 20 µg/mL myokinase. Incubations were carried out at 37°C for 10 minutes and terminated by addition of 1 mL of a solution containing 100 µg ATP, 50 µg cAMP, and 15 000 cpm [³H] cAMP. Cells were pelleted by centrifugation at 300g for 5 minutes. cAMP was isolated from the supernatant by sequential Dowex and alumina chromatography and was corrected for recovery with [³H] cAMP as the internal standard. Adenyl cyclase activity was linear with time and cell number over the ranges used.

ß-Adrenoceptor stimulation of adenyl cyclase activity was assessed by isoproterenol (100 µmol/L) in the presence of GTP (100 µmol/L). G protein–selective stimulation was assessed by the addition of NaF (20 mmol/L) and AlCl₃ (20 µmol/L), which complexes to form the active species aluminum fluoride (AlF₄). Maximal catalytic activity was assessed with forskolin (10 µmol/L). Aluminum fluoride and isoproterenol-stimulated activities were expressed relative to maximal forskolin-stimulated activity. This proportional method of expression was selected prospectively and is consistent with that used in our previous studies comparing maximal adenyl cyclase activity in subject groups.^{9 27 28} This approach was chosen on the basis of two major considerations. First, although absolute levels of activity (ie, as expressed as pmol/min) may differ between lymphocyte subpopulations, relative stimulation is comparable.^{29 30} Second, in preliminary studies, we determined that the coefficient of variation is lower when stimulated enzyme activity is expressed relative to forskolin-stimulated activity rather than basal activity (as we have done previously) or when expressed as absolute activity.

Data Analysis
Because relative proportions are log normally distributed,³¹ G protein data are expressed as the geometric means±average geometric SEM. For toxin-mediated labeling, one-group t tests were performed to assess differences in proportional labeling. For immunoblotting studies, two-group t tests were performed. For studies of adenyl cyclase activity, ANOVAs were performed initially, followed by group comparisons as appropriate. A value of P<.05 on a two-tailed test was considered significant.

	Results

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No significant alterations in overnight sodium excretion were detected between older hypertensive subjects or younger normotensive control subjects (the Table). No significant differences in blood pressure were apparent between older and younger control subjects (the Table). Normotensive and hypertensive subjects were comparably aged (the Table). Body mass did not differ between the three groups (the Table).

Alterations in Adenyl Cyclase Activity
In lymphocytes from both younger hypertensive and older normotensive subjects, isoproterenol-stimulated adenyl cyclase activity was significantly reduced (compared with the activity in lymphocytes from younger normotensive control subjects, Fig 1, top). However, whereas in hypertensive subjects, NaF/AlCl₃–stimulated activity was impaired, it was unaffected in older subjects (Fig 1, bottom). Aluminum fluoride–stimulated activity was inversely correlated with mean arterial blood pressure both when including only younger normotensive subjects and hypertensive subjects (r=-.51, P=.044) and also when including older subjects (r=-.51, P=.014). Forskolin-stimulated activities did not differ between the three groups (younger normotensive subjects, 57±5 pmol · min^-1 · 10⁶ cells; younger hypertensive subjects, 65±5 pmol · min^-1 · 10⁶ cells; older normotensive subjects, 62±6 pmol · min^-1 · 10⁶ cells).

View larger version (34K): [in this window] [in a new window]   Figure 1. Alterations in lymphocyte adenyl cyclase activity with hypertension and aging. Top, Alterations in isoproterenol-stimulated activity. Bottom, Alterations in aluminum fluoride–stimulated activity. Data represent mean±SEM. *P<.05 vs younger normotensive subjects based on ANOVA, followed by t tests as appropriate.

Alterations in CT-Mediated Labeling
In control studies, autoradiographs of SDS-PAGE with mononuclear leukocyte lysates incubated with CT and [³²P]NAD demonstrated specific labeling of a single band at approximately 42 to 43 kD (Fig 2). No qualitative alterations in mobility of specific CT-mediated labeling were apparent in autoradiographs from either hypertensive subjects or older subjects. However, the extent of CT-specific labeling was decreased in lymphocytes from hypertensive subjects to 63±10% of labeling in young normotensive subjects (P<.05, Figs 2 and 3). In contrast, no significant alterations in the extent of CT labeling were evident in older normotensive subjects (Fig 3).

View larger version (25K): [in this window] [in a new window]   Figure 2. Alterations in cholera toxin–mediated ADP ribosylation in mononuclear leukocytes from hypertensive subjects. A, Autoradiograph of an SDS-PAGE from a representative normotensive subject vs hypertensive subject study. (+) indicates lanes with lysates incubated with [32P]NAD and cholera toxin; (-), lanes with lysates incubated with [32P]NAD alone. Arrows represent migration of molecular weight markers. Toxin-specific labeling of a 42- to 43-kD peptide is evident, which is reduced in the hypertensive subject. B, Corresponding densitometric tracing of toxin-specific labeling in samples from the normotensive subject (solid line) and the hypertensive subject (broken line).

View larger version (11K): [in this window] [in a new window]   Figure 3. Alterations in toxin-mediated labeling of G proteins in mononuclear leukocytes from hypertensive (n=7) and older subjects (n=13) with pertussis toxin–mediated labeling (Gi/Go) and cholera toxin-mediated labeling (Gs). *P<.05 vs normotensive, younger controls.

Alterations in PT-Mediated Labeling
Autoradiographs of SDS-PAGE with lysates incubated with PT and [³²P]NAD demonstrated specific labeling of a 39-kD peptide. Labeling of a 41-kD peptide was seen inconsistently and was predominantly not toxin-dependent. In lymphocytes from older subjects, PT-specific labeling of the 39-kD peptide was increased to 158±18% of the labeling seen in lymphocytes from younger subjects (P<.05, Figs 3 and 4). In contrast, PT-specific labeling was not significantly altered in lymphocytes from hypertensive subjects (Fig 3).

View larger version (28K): [in this window] [in a new window]   Figure 4. Alterations in pertussis toxin–mediated ADP ribosylation in mononuclear leukocytes from older subjects. A, Autoradiograph of an SDS-PAGE from a representative younger vs older subject study. (+) indicates lanes with lysates incubated with [32P]NAD and pertussis toxin; (-), lanes with lysates incubated with [32P]NAD alone. Arrows represent migration of molecular weight markers. Toxin-specific labeling of a 39-kD peptide is evident, which is increased in the older subject (labeling of a 41-kD peptide was seen only inconsistently). B, Corresponding densitometric tracing of toxin-specific labeling in samples from the younger subject (solid line) and the older subject (broken line).

Immunochemical Measurement of G_s: Alterations With Hypertension
To determine whether the decrease in CT-mediated labeling in lymphocytes from hypertensive subjects represented an immunodetectable decrease in the -subunit of G_s, Western blots were performed using the _s-specific antisera RM/1 (DuPont–New England Nuclear). On Western blots of mononuclear leukocytes, this antisera detected specific labeling of a 42- to 43-kD peptide (migrating similarly to the peptide identified by CT-mediated labeling techniques). This corresponded to the antisera-specific labeling of a 45-kD peptide in the bovine brain standard (the predominant _s species in this preparation; Fig 5). In contrast to the findings of reduced CT-mediated labeling in lymphocytes from hypertensive subjects, Western blot determinations demonstrated that _s expression, assessed by immunodetection, was not decreased and in fact demonstrated a borderline increase (Fig 6).*

View larger version (92K): [in this window] [in a new window]   Figure 5. Autoradiograph of a Western blot membrane developed with the enhanced chemiluminescence system, showing s labeling of immunoblots. s-Specific antisera (RM/l) was used to identify a 42- to 43-kD peptide in lymphocytes from younger normotensive subjects (YN) as compared to a bovine brain G protein standard (STD) which predominantly expresses a 45-kD s.

View larger version (16K): [in this window] [in a new window]   Figure 6. Alterations in immunochemical s- and ß-subunit labeling in lymphocytes from hypertensive subjects. Labeling in lymphocytes from hypertensive subjects is expressed as a proportion of the average labeling in lymphocytes from normotensive subjects.

Immunochemical labeling of lymphocyte ß-subunits (with SW/1 antisera) was not significantly altered in borderline-hypertensive subjects (Fig 6).

Immunochemical Measurement of G_i: Alterations With Aging
To determine whether the increase in PT-mediated labeling represented an increase in _i- or _o-subunit labeling, Western blots were performed using the _i,1,2-specific antisera AS/7, the _i3-specific antisera EC/2, and the _o-specific antisera GC/2. Neither _o nor _i3 labeling was detected in mononuclear leukocyte preparations although evident in concurrently assayed bovine brain standards (data not shown). In contrast, Western blots of mononuclear leukocytes using the _i1,2-specific antisera identified a 39-kD peptide (Fig 7) corresponding to the major peptide labeled using PT and [³²P]NAD. When mononuclear leukocyte _i labeling in younger and older subjects was compared, no significant alterations were apparent (Fig 8). ß-Subunit labeling was also not different between older and younger subjects (Fig 8).

View larger version (77K): [in this window] [in a new window]   Figure 7. Autoradiograph of a Western blot membrane developed with the enhanced chemiluminescence system, showing i labeling of immunoblots. il,2-Specific antisera (AS/7) was used to identify a 39-kD peptide in lymphocytes from younger normotensive subjects (YN) compared with a bovine brain G protein standard (STD) that predominantly expresses a 40- and 41-kD i.

View larger version (15K): [in this window] [in a new window]   Figure 8. Alterations in immunochemical i- and ß-subunit labeling in lymphocytes from older subjects. Labeling in lymphocytes from older subjects is expressed as a proportion of the average labeling in lymphocytes from younger subjects.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study demonstrates that in human mononuclear leukocytes, ß-adrenergic–stimulated adenyl cyclase activity is comparably impaired in older and in hypertensive subjects, whereas G protein–stimulated activity (as assessed with aluminum fluoride) is impaired in hypertensive but not in older subjects. Furthermore, we demonstrated that CT and PT mediate specific labeling of 42- and 43-kD peptides and 39-kD peptides, respectively, and that the extent of labeling is altered in hypertension and aging. Specifically, CT-mediated labeling is reduced in mononuclear leukocytes from hypertensive subjects and PT-mediated labeling is increased in mononuclear leukocytes from older subjects. However, these alterations are independent of any alterations in - or ß-G protein subunits as assessed immunochemically.

CT-specific [³²P]NAD labeling as well as immunochemical labeling with _s-specific antisera identified a human lymphocyte protein with a molecular weight of approximately 42 to 43 kD. Consistent with the findings of previous investigators,^{32 33} we conclude this represents labeling of the -subunit of the guanine nucleotide regulatory protein of stimulation (G_s).

PT-specific labeling and immunochemical labeling with _i1,2-specific antisera identified a 39-kD protein consistent with labeling of inhibitory guanine nucleotide regulatory proteins (G_i1 or G_i2).³³ Although [³²P]NAD labeling with PT also identified labeling of a 41-kD peptide, this was predominantly not toxin-specific and was not identified with _i1,2-, _i3-, or _o-specific antisera. The identity of this peptide is yet to be determined. Neither G_i3 nor G_o was detected in lymphocytes.

The reduction in CT-mediated labeling in younger hypertensive subjects parallels the reduction in mononuclear leukocyte adenyl cyclase activation reported in the present study and previously reported by ourselves and others (as reviewed in Reference 16¹⁶ ). In addition, a recent study demonstrated reduced CT-stimulated cAMP in lymphocytes from hypertensive subjects.³⁴ Our current findings are consistent with either decreased expression or bioactivity of the -subunit of G_s. However, decreased mononuclear leukocyte CT-mediated labeling in hypertensive subjects was not accompanied by any reduction in expression of the - or ß-subunits of the stimulatory G protein G_s as assessed immunochemically. An alteration in CT-mediated ADP ribosylation of the -subunit of G_s may reflect other factors in addition to an alteration in the mass of _s and as such has been suggested to be a "quasifunctional" measure of G_s activity.³⁵ The extent of CT labeling is dependent on the kinetics of the dissociation of - and ß/-subunits and is proportional to the ratio of - to ß/-subunits. Notably, ß-subunit concentration was marginally increased in hypertensive subjects. However, the relative ratio of the concentration of _s- to ß-subunits (determined immunochemically) was not altered. Posttranslational modifications of either the -subunit³⁶ or the -subunit³⁷ have been associated with alterations in G protein function and specifically G protein–receptor coupling.³⁸ Whether these modifications alter CT-mediated labeling is unclear. Also, the alterations in CT-mediated labeling could reflect alterations in endogenous levels of G protein ADP ribosylation.³⁵ An alteration in NAD⁺ nucleosidase activity could result in altered toxin-mediated labeling.^{35 39} However, the selectivity of the effects seen (reduced CT labeling without alterations in PT labeling) would make this latter explanation unlikely.

An alteration in human lymphocyte G_s labeling has not been demonstrated previously in human hypertension. However, alterations in G_s protein function and expression have been reported in rat models of hypertension, in which receptor-stimulated adenyl cyclase activation also has been reported to be impaired (reviewed in Reference 40⁴⁰ ). A reduction in vascular G_s function in spontaneously hypertensive rats (SHR) has been suggested based on isolated organ bath studies.⁴¹ Furthermore, a reduction in G_s levels has been reported in studies of vascular tissue from Milan hypertensive rats⁴² and renal tissue from SHR⁴³ as assessed by immunoblots. In contrast, several investigators have reported no significant alterations in either cardiac or vascular G_s in SHR as assessed by immunoblots^{44 45 46} or by toxin-mediated labeling.⁴⁶

PT-mediated labeling was not altered in lymphocytes from hypertensive subjects. Alterations in G_i concentrations have been reported in rat models of hypertension. However, these alterations appear to be variable, dependent both on the model as well as the tissues studied. Thus, in one-kidney/one clip, deoxycorticosterone-treated, reduced renal mass rats⁴⁷ and Dahl salt-sensitive rats,⁴⁸ increased cardiac G_i concentrations have been reported. In contrast, in Milan hypertensive rats, vascular G_i levels are reportedly unchanged.⁴² In SHR, cardiac and vascular G_i levels have been reported to be either increased^{46 49} or unchanged,^{44 45} whereas platelet G_i levels have been reported to be decreased.⁵⁰

Platelet G proteins have been examined in hypertensive subjects.⁵¹ No alterations in immunoblots of G proteins (G_i or G_s) were reported. However, in platelets from hypertensive subjects, receptor-stimulated adenyl cyclase activity was reported to be increased. This diverges from the attenuation of both ß-adrenergic–mediated vasodilator response and lymphocyte receptor–stimulated adenyl cyclase activation generally (but not universally) reported in studies of hypertensive subjects.^{1 8 9 16 20} Taken together, these findings are consistent with the notion that regulation of human platelet adenyl cyclase activation (and G protein regulation) differs from that of vascular smooth muscle and nucleated circulating cells.

With aging, a reduction in the extent of lymphocyte adenyl cyclase activation by stimulators acting both at the level of the receptor and distal to the receptor have been described previously (reviewed in Reference 18¹⁸ ). In the present study, we reconfirmed the reduction in ß-adrenergic–stimulated adenyl cyclase activity. However, aluminum fluoride–stimulated activity was not reduced in lymphocytes from older subjects. This differential impairment in adenyl cyclase activity does not parallel the pattern seen in lymphocytes from hypertensive subjects. This might suggest that different pathological mechanisms account for the alterations in ß-adrenergic–mediated responses seen in these groups. In small studies such as ours, we cannot rule out the possibility of a type 2 error for failing to detect reductions in aluminum fluoride–stimulated activity in older subjects comparable to those seen in hypertensive subjects. However, it is notable that the inverse relationship between aluminum fluoride–stimulated adenyl cyclase activity and blood pressure remained even when the older subjects were included. Furthermore, in crude preparations, it has been suggested that aluminum fluoride–stimulated activity might be a more selective index of G_s activity (compared with the effects of hydrolysis-resistant GTP analogues).⁵² If so, this would be consistent with our finding that both aluminum fluoride–stimulated adenyl cyclase activity and G_s toxin labeling were altered in lymphocytes from hypertensive subjects but not in lymphoctyes from older, normotensive subjects.

PT-mediated labeling was increased in lymphocytes from older subjects. However, immunochemically assessed _i concentration was not altered. The increase in PT-mediated labeling is consistent with results reported recently in a study of lymphocyte G proteins in elderly subjects⁵³ and parallels an age-related increase in rat myocardial G_i.⁵⁴ Notably, a reduction in a myocardial G_s with aging has also been reported.⁵⁵ In the absence of any alterations in _i- or ß-subunit concentrations, the increase in PT-mediated labeling could reflect decreased endogenous ADP ribosylation and/or alterations in the kinetics of association of _i- and ß/-subunits. As noted above, an alteration in NAD⁺ nucleosidase activity is unlikely to explain the selective alterations in PT-mediated labeling in older subjects. This discordance between the findings of increased PT substrate but unaltered G_i levels (as assessed immunochemically) parallels the situation seen in cardiac tissues in human heart failure as reported by some⁵⁶ but not all investigators (reviewed in Reference 57⁵⁷ ).

Finally, it should be recalled that other G proteins can serve as PT substrates. Could increased expression of another G protein with a mobility on SDS-PAGE comparable to G_i account for our findings? PT also mediates ADP ribosylation of the -subunit of G_o.¹¹ However, we were unable to detect expression of G_o in human lymphocytes.

The alterations in G_s proteins suggested by the toxin-mediated labeling studies in lymphocytes from hypertensive subjects are consistent with the impairment in aluminum fluoride–stimulation of adenyl cyclase activity. However, whether the increase in G_i labeling seen in lymphocytes from older subjects can be implicated in the impairment in ß-adrenoceptor–stimulated adenyl cyclase activity is less clear. In vitro models have suggested that increased expression of inhibitory G proteins may be associated with tonic inhibition of adenyl cyclase activation. Specifically, norepinephrine-induced heterologous desensitization of adenyl cyclase stimulation has been linked to an increase of inhibitory G protein function.¹⁴ Also, attenuation of inhibitory G protein activity (using PT) results in enhanced stimulation of adenyl cyclase in a number of models.^{12 15} Increased G_i function has been identified in several models of heart failure and has been linked to impaired stimulation of adenyl cyclase in that disease (reviewed in Reference 57⁵⁷ ). However, other alterations both at the level of the receptor and the catalytic subunit may also be important in heart failure. Furthermore, increased G_i1 and G_i2 proteins in adipocytes from aged rats have been associated with a parallel reduction in the sensitivity of adenyl cyclase to stimulation with isoproterenol.⁵⁸

In summary, the present study suggests important alterations in the function but not the expression of G proteins in hypertension and in aging. In both hypertension and aging, ß-adrenoceptor–mediated stimulation of adenyl cyclase is impaired. However, these conditions appear to be associated with divergent alterations in stimulatory and inhibitory G proteins.

	Acknowledgments

 
This work was supported by a grant-in-aid from the Medical Research Council of Canada. Dr Feldman is supported by a Career Investigator Award from the Heart and Stroke Foundation of Ontario.

	Footnotes

 
¹ The defect in lymphocyte ß-adrenergic responsiveness may be predominantly characteristic of younger, white hypertensive subjects.¹⁹

² In contrast, regulation of lymphocyte ß-adrenergic adenyl cyclase activation does not parallel regulation of cardiac ß₁-adrenergic responsiveness or ß₁-adrenoceptors in other tissues.

³ In comparing the results from Western blots and toxin-labeling studies, we considered whether differences between techniques in the processing of preparations could account for the difference in results. Notably, after incubation of lymphocyte lysates with toxin-[³²P]NAD, samples were centrifuged and resuspended to reduce nonspecific labeling before solubilization. This approach might result in selective loss of a "sequestered" (non–membrane associated) population of _s-subunits. Thus, an increased proportion of sequestered G_s in lymphocytes from hypertensive subjects could have accounted for a decrease in CT-mediated labeling in washed lysates that might not be reflected in preparations using whole-cell pellets that were directly solubilized, as in the Western blot studies. Thus, on two occasions, immunoblotting of _s proteins was performed using a lysate preparation identical to that used in CT-labeling studies. With this technique, _s labeling by immunodetection was not decreased in mononuclear leukocytes from hypertensive subjects (data not shown).

Received July 18, 1994; first decision August 31, 1994; accepted August 7, 1995.

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