Introduction

Abstract Introduction Methods Results Discussion References

Diabetes mellitus is associated with an increased incidence of cardiovascular disease (Garcia et al., 1974), which has been suggested to be due, at least in part, to an increased pressor response to NA and other circulating hormones (Christlieb et al., 1976). Studies in chronic, chemically induced models of diabetes in animals have yielded conflicting results, although many investigators have reported increases in the maximum contractile responses to alpha adrenoceptor stimulation (Brody and Dixon, 1964; MacLeod, 1985; Mulhern and Docherty, 1989; White and Carrier, 1988). Other investigators have reported increases in sensitivity to alpha adrenoceptor stimulation with no change in the maximum responses (Cohen et al., 1990) or decreases in the maximum responses (Pfaffman et al., 1982) of arteries from animals with chronic, chemically induced diabetes. The reason for these differences is not entirely clear, but contributing factors may be differences in the species, duration of diabetes and vascular preparation studied. This laboratory has consistently shown that maximum contractile responses to NA of aorta, mesenteric and caudal arteries from rats with STZ-induced diabetes of 12 to 14 weeks duration are increased, with little or no increase in the sensitivity (pD₂ or log EC₅₀) compared with responses of arteries from age-matched control rats (Abebe et al., 1990, 1994; MacLeod, 1985; Weber et al., 1996). The enhanced contractile responsiveness to NA is not due to dysfunctional endothelium (Harris and MacLeod, 1988; Weber et al., 1996), adrenergic neuropathy (Weber and MacLeod, 1994) or a generalized increase in contractility (Abebe et al., 1994; MacLeod, 1985; Weber and MacLeod, 1994) and was shown to be selectively mediated by alpha-1 adrenoceptors, not alpha-2 adrenoceptors, based on pA₂ values for prazosin and yohimbine (Abebe et al., 1990). Furthermore, we have demonstrated that bypassing the receptors by directly stimulating G proteins with aluminum fluoride resulted in a similarly enhanced contractile response in aorta, mesenteric and caudal arteries (Weber et al., 1996).

The alpha-1 adrenoceptor is thought to play the predominant role in mediating NA-stimulated vasoconstriction, although alpha-2 adrenoceptors may contribute, particularly in small arteries such as the caudal artery. However, we have found that in membranes prepared from endothelium-denuded aorta and caudal arteries from normal rats, NA mediates increases in high-affinity GTPase activity (a measure of G protein stimulation) exclusively via the alpha-1 adrenoceptor (Weber and MacLeod, 1996). For this reason and because the contractility studies suggested the alpha-1 adrenoceptor mediates the enhanced contractile response of diabetic arteries to NA, we examined only the alpha-1 adrenoceptor in the present study.

The alpha-1 adrenoceptor is thought to be coupled to two different signal transduction pathways (reviewed in Minneman, 1988). The first pathway may involve coupling of the alpha-1 adrenoceptor to a G protein that is sensitive to PTX (Liebau et al., 1989). In support of this, the presence of PTX-sensitive G proteins, G_i2 and/or G_i3, has been confirmed in immunoblots of rat aorta and caudal artery membranes (Weber and MacLeod, 1996). Although the mechanism is not known, G_i stimulated by the alpha-1 adrenoceptor has been suggested to be involved in opening of calcium channels in vascular smooth muscle (Liebau et al., 1989). The second pathway that couples to the alpha-1 adrenoceptor in vascular smooth muscle is PLC via a G protein that is insensitive to PTX (Liebau et al., 1989; Minneman, 1988). Because G_q/11 has been detected in Western blots of rat aorta and caudal artery membranes (Weber and MacLeod, 1996) and has been shown to couple to PLC in a PTX-insensitive manner in other tissues (Shenker et al., 1991), it seems quite likely that G_q/11 is the G protein coupling the alpha-1 adrenoceptor to PLC in vascular smooth muscle. PLC catalyzes the hydrolysis of PIP₂ to form two second messengers: Ins(1,4,5)P₃, which causes release of intracellular calcium, and DAG, which activates protein kinase C (Minneman, 1988).

Studies in this laboratory have shown an enhanced breakdown of PIP₂, formation of total inositol phosphates, formation of phosphatidic acid from DAG and production of Ins(1,4,5)P₃ in aorta and mesenteric arteries from STZ-diabetic rats compared with arteries from control rats in response to NA (Abebe and MacLeod, 1991a, 1991b, 1992). We have also found that the enhanced contraction elicited in arteries from diabetic rats by alpha-1 adrenoceptor stimulation or exposure to fluoroaluminates relies on intracellular calcium release, influx of calcium and PKC activity to a larger but proportionately similar extent as that of arteries from control rats (Abebe et al., 1990., 1994; Weber et al., 1996). These findings imply that all of the second-messenger mechanisms activated by the alpha-1 adrenoceptor are increased simultaneously to mediate the enhanced diabetic contraction. A change at a point both proximal and common to stimulation of PLC and calcium channels but distal to the alpha-1 adrenoceptor, such as increased number or activity of alpha-1 adrenoceptor-associated G proteins, seems most probable to mediate the enhanced responses in diabetic arteries.

The purpose of the present investigation was, first, to confirm through direct measurement that alpha-1 adrenoceptors were not changed in arteries from 12- to 14-week STZ-diabetic rats compared with those of age-matched control rats. Second, because recent evidence suggests that an increase in the number of G proteins or the efficiency of their coupling with receptors could lead to an increase in maximum response (reviewed in Raymond, 1995), we wanted to determine whether changes in the interaction between alpha-1 adrenoceptors and their G proteins or in the levels of these G proteins could account for the enhanced responsiveness of arteries from diabetic rats. The number and affinity of alpha-1 adrenoceptors were determined from [³H]prazosin saturation binding in membranes prepared from endothelium-denuded aorta and caudal arteries from control and diabetic rats. The ability of the GTP analog Gpp(NH)p to shift the affinity of receptors for agonists from high to low has been used as an index of the interaction of receptors with G proteins (Jagadeesh and Deth, 1987). Therefore, the ability of Gpp(NH)p to shift the affinity of the alpha-1 adrenoceptor for NA was determined in competition binding experiments with [³H]prazosin in membranes prepared from aorta and caudal artery from control and diabetic rats. The levels of G proteins were also determined in control and diabetic rat artery membranes by performing Western blots and densitometry using G protein subtype-selective antisera. Finally, ouabain-inhibitable Na⁺/K⁺-ATPase activity was used to determine any differences in purity of the membranes between control and diabetic artery preparations.

	Methods

Abstract Introduction Methods Results Discussion References

Control and diabetic rats. Male Wistar rats (170-230 g) obtained from Charles River (Montreal, Quebec, Canada) or U.B.C. Animal Care Unit (Vancouver, British Columbia, Canada) were housed and treated according to the guidelines of the Canadian Council on Animal Care. Rats were given a single intravenous injection of STZ (60 mg_/kg) or the citrate buffer vehicle while under light halothane anesthesia. STZ-treated rats with a blood glucose of >10 mmol/l (measured using an Ames glucometer) at 1 week after injection were considered to be diabetic and were retained for experiments. Control and diabetic rats were housed separately and given free access to food and water. At 12 to 14 weeks after STZ or vehicle injection, animals were weighed and injected with an overdose of pentobarbital (65 mg/kg intraperitoneal). Blood was collected from the open chest with a heparin-rinsed Pasteur pipette, placed into a microcentrifuge tube with heparin and then centrifuged in an Eppendorf microcentrifuge for 10 min at 4°C. The plasma was separated from the packed red cells with a Pasteur pipette, divided into aliquots and stored at 30°C until assay for insulin and glucose. The aorta and caudal arteries were removed and cleaned of connective tissue. Endothelium was removed by passing a wire through the lumen of each artery and gently rotating the artery over the wire. Then, each artery was frozen with clamps cooled in liquid nitrogen and stored at 70°C until use.

Insulin and glucose assay. Plasma was assayed for glucose using the Peridochrom (Boehringer-Mannheim, Laval, Quebec, Canada) glucose assay kit, which is a colorimetric assay based on glucose oxidation by glucose-6-phosphate dehydrogenase. Rat plasma insulin levels were measured using Linco (Linco Inc., St. Charles, MO) kits, radioimmunoassays that use anti-rat insulin antibody and rat insulin standards.

Preparation of membrane. Aliquots of the same crude membrane preparations were used for the receptor binding assays, Western blots and Na⁺/K⁺-ATPase assay. Briefly, 18 aortas or 15 caudal arteries (previously cleaned and frozen and intact except for the absence of endothelium) from control or diabetic rats were pooled. The frozen arteries were quickly weighed and placed in 5 ml of ice-cold homogenization buffer (final composition: 20 mM Tris·HCl, 1 mM dithiothrietol, 1 mM EDTA, 100 µg/ml trypsin inhibitor, 1 mM phenylmethylsulfonyl fluoride, 3 mM benzamidine HCl, 1 µM leupeptin and 1 µM pepstatin A, pH 8.0). Arteries were minced with scissors and then homogenized on ice with a hand-held Omni 2000 homogenizer at the highest setting with four 10-sec bursts. The homogenizer blade was washed with four 1.25-ml aliquots of homogenization buffer, and the washings were added to the homogenate to give a final volume of 10 ml. The homogenate was then centrifuged at 900 × g for 10 min at 4°C, and the resulting PNS was collected. An aliquot of the PNS was taken for protein and Na⁺/K⁺-ATPase assay, and the remainder was centrifuged at 105,000 × g for 1 hr at 4°C. The resulting crude membrane pellet was resuspended in 1 ml of 50 mM triethanolamine and divided into aliquots. One aliquot was taken for measurement of protein, and the remaining aliquots were frozen at 70°C until used in the other assays. Protein contents of the PNS and the resuspended membrane pellet were measured using the BioRad (Hercules, CA) protein dye binding assay system. Bovine serum albumin was used as a standard. Neither the starting weights of each frozen artery pool [2.1 ± 0.2 and 1.8 ± 0.1 g, respectively, for control and diabetic aorta (n = 9) and 1.0 ± 0.1 and 0.9 ± 0.1 g, respectively, for control and diabetic caudal arteries (n = 10)] nor the yield of protein in the final membrane preparations [0.58 ± 0.07 and 0.74 ± 0.07 mg, respectively for control and diabetic aorta (n = 9) and 1.12 ± 0.10 and 1.39 ± 0.10 mg, respectively, for control and diabetic caudal arteries (n = 10)] differed between control and diabetic arteries.

Na⁺/K⁺-ATPase assay. The method for measuring ouabain-inhibitable Na⁺/K⁺-ATPase activity, based on the measurement of free ³²P_i from [³²P]ATP, was as performed previously in this laboratory (Weber and MacLeod, 1996), except that 150 mM sodium azide was used.

[³H]Prazosin binding assays. Binding of [³H]prazosin (79.8 Ci/mmol) was determined using a modification of the methods of Cheung and Triggle (1988). Saturation binding assays were carried out in duplicate using aorta and caudal artery membrane (8-20 µg protein/tube) in a total reaction volume of 250 µl (final binding buffer composition: 50 mM triethanolamine HCl, 5 mM MgCl₂, 1 mM EGTA and 0.1% ascorbic acid, pH 7.4). The binding assay was started by the addition of membrane after warming the tubes for 2 min and allowed to proceed for 20 or 30 min at room temperature. Binding was stopped by the addition of 2 ml of ice-cold assay buffer and rapid filtration over GF/C filters under a vacuum. The filters were washed with an additional four 2-ml aliquots of ice-cold assay buffer, placed in vials and dispersed in scintillant with shaking for several hours before scintillation counting. Specific binding was determined at each [³H]prazosin concentration by subtracting the binding remaining in the presence of 10 µM phentolamine and was 57% (aorta) or 73% (caudal artery) of total binding at 0.5 to 1.5 nM [³H]prazosin. Counting efficiency of tritium was 23% at 0.5 nM, 30% at 1.0 nM and 45% at 16 nM [³H]prazosin. Preliminary experiments had shown that steady-state [³H]prazosin binding was reached by 20 min under these assay conditions (data not shown). Also, the levels of membrane protein used in this assay were determined in preliminary experiments to be on the linear portion of the protein-dependence curve (data not shown).

Western blotting and densitometry. Proteins in control and diabetic aorta and caudal artery membranes were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as previously performed in this laboratory (Weber and MacLeod, 1996). Specific anti-G antisera, AS/7, EC/2 or QL, and ECL were used for detection. Integrated absorbance was measured using a Visage densitometer. A human platelet internal standard (47 µg of protein/lane) was also quantified for immunoreactivity for each antibody and each blot and was used to correct each control and diabetic sample. The integrated absorbance values for each sample were also corrected for the amount of membrane protein loaded. In preliminary experiments, the levels of artery membrane protein used (2-13 µg protein/lane) were found to be on the linear portion of the absorbance-vs.-protein level curve (data not shown).

Materials. Film, horseradish peroxidase-conjugated donkey anti-rabbit antibody, ECL reagent kits and [³²P]ATP (3000 Ci/mmol) were obtained from Amersham (Oakville, Ontario, Canada). 2-Mercaptoethanol, acrylamide, ammonium persulfate, dithiothrietol, glycine, N,N-methylene-bisacrylamide, N,N,N,N-tetraethylmethylene-diamine, nitrocellulose paper and sodium dodecyl sulphate were obtained from BioRad (Hercules, CA). Anti-G_i1,2 (AS/7) antiserum, anti-G_i3/o (EC/2) antiserum, anti-G_q/11 (QL) antiserum and [³H]prazosin (80 Ci/mmol) were obtained from Dupont NEN (Mississauga, Ontario, Canada). Ouabain was obtained from ICN Biomedicals (Aurora, OH). Sodium pentobarbital was obtained from MTC Pharmaceuticals (Cambridge, Ontario, Canada). All other chemicals and reagents were obtained from Sigma Chemical (St. Louis, MO).

Data analysis and statistics. The results shown are from different membrane preparations (n). All results are expressed as mean ± S.E.M. One- or two-way ANOVAs, followed by Bonferroni's post hoc tests as needed, were used in comparisons of the data. Saturation binding data were analyzed using GraphPAD Prism software (San Diego, CA) and used to generate estimates of K_d and B_max. For the NA competition curves, a best-fit comparison was made between nonlinear one- and two-site competition binding. In GraphPAD Prism, this comparison is made by calculating an F test of the sum-of-squares of the two fits. Data for all nonlinear fitting were displayed on individual curves, with the individual estimates used for statistical purposes. The IC₅₀ values calculated from the NA competition curves were used for all statistical comparisons and to generate estimates of K_i using the Cheng and Prusoff (1973) equation as follows: where R is the concentration of [³H]prazosin used in the experiment, and K_d is the experimentally determined value from [³H]prazosin saturation curves.

	Results

Abstract Introduction Methods Results Discussion References

Animals and membrane preparations. The arteries used for the present study were obtained from 12- to 14-week STZ-diabetic rats and age-matched controls. The STZ-treated rats displayed the typical complications of diabetes such as polyuria, polydipsia, hyperphagia, emaciation and cataracts. The weight of the diabetic rats used in the experiments was 389 ± 6 g (mean ± S.E.M.; n = 116 rats) at the time of death, which was significantly (P < .05; one-way ANOVA) less than that of the age-matched control rats (480 ± 4 g; n = 110). The diabetic rats were significantly (P < .05; one-way ANOVA) hypoinsulinemic, with plasma insulin levels of 1.43 ± 0.15 ng/ml compared with control levels of 6.41 ± 0.30 ng/ml. Finally, the plasma glucose levels of the diabetic rats, 24.48 ± 0.76 mM, were significantly (P < .05; one-way ANOVA) higher than the value of 9.46 ± 0.42 mM determined for the control rats.

Alpha-1 adrenoceptor saturation binding. In [³H]prazosin saturation assays, a single site was found to which binding was saturable and which corresponded to the alpha-1 adrenoceptor in its affinity for prazosin, in both aorta and caudal artery membranes (fig. 1). In both aorta and caudal artery membranes, we found no difference between control and diabetic rats in the K_d value, which reflects receptor affinity for [³H]prazosin (table 2). We also found no difference between control and diabetic aorta membranes in the B_max value for [³H]prazosin, which reflects the alpha-1 adrenoceptor number (table 2). However, in caudal artery membranes prepared from diabetic rats, the B_max value was significantly less than the B_max value obtained in control caudal artery membranes (table 2). The statistical findings were the same regardless of whether the [³H]prazosin binding data was expressed corrected for membrane protein or for the sodium pump activity of the membrane preparation (table 2).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Representative [3H]prazosin saturation curves in aorta and caudal artery membranes from control () and diabetic () rats. Mean values of several determinations are shown.

Alpha-1 adrenoceptor competition curves with NA in the absence of sodium chloride. A second series of [³H]prazosin binding studies were performed to investigate competition with NA in the presence or absence of the nonhydrolyzable GTP analog, Gpp(NH)p (0.1 mM). This series of experiments was performed in the absence of monovalent cations. Theoretically, under experimental conditions in which GTP or an analog is absent, two agonist binding sites can be detected on receptors: a high-affinity and a low-affinity site. When GTP or Gpp(NH)p is added, agonists bind to a single low-affinity site. In membranes prepared from both aorta and caudal artery from control rats, the binding of NA to the alpha-1 adrenoceptor in the absence of Gpp(NH)p was best fit to a two-site model (fig. 2; P < .05 for two-site over one-site in an F test) with a high- and a low-affinity binding site (table 3). The fraction of receptors in the high-affinity state was estimated to be 35 ± 8% in control aorta and 28 ± 5% in control caudal arteries membranes in the absence of Gpp(NH)p. As expected, in the presence of Gpp(NH)p, only one low-affinity site was detected in control aorta and caudal artery membranes (fig. 2, table 3). However, in membranes prepared from diabetic aorta and caudal artery, only one low-affinity site was detected, regardless of whether Gpp(NH)p was present (fig. 2, table 3).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Representative competition curves of [3H]prazosin with NA in membranes prepared from control and diabetic caudal arteries in the absence () and presence () of 0.1 mM Gpp(NH)p. This series of experiments was run in the absence of NaCl. Mean values of several determinations shown. Similar results were obtained in aorta from control and diabetic rats.

Alpha-1 adrenoceptor competition curves with NA in the presence of sodium chloride. A third series of [³H]prazosin binding experiments looked at the competition with NA as in the previous series, except in the presence of 200 mM NaCl. This was prompted by the observation of Cheung and Triggle (1988) that monovalent cations alone, in the absence of Gpp(NH)p, can reduce the affinity of the alpha-1 adrenoceptor for agonists. In the control and diabetic aorta and caudal artery membranes studied, competition by NA for [³H]prazosin binding was best fit to a one-site model (P = 1.0; F test for two-site over one-site), regardless of whether Gpp(NH)p was present (fig. 3, table 4). The IC₅₀ of that one site in the absence of Gpp(NH)p in both aorta and caudal artery membranes resembled that of the low-affinity site detected when NaCl was not present in the binding buffer (table 4 vs. table 3). In this series of experiments with NaCl, in aorta preparations the IC₅₀ of the site did not differ significantly between control and diabetic or between the absence and presence of Gpp(NH)p (table 4). However, in caudal artery, there was a further significant decrease in the NA IC₅₀ in the presence of Gpp(NH)p in both control and diabetic preparations. No difference in the agonist IC₅₀ values was detected between the control and diabetic caudal artery preparations in the absence or presence of Gpp(NH)p (table 4).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Representative competition curves of [3H]prazosin with NA in membranes prepared from control and diabetic caudal arteries in the absence () and presence () of 0.1 mM Gpp(NH)p. This series of experiments was run in the presence of 200 mM NaCl. Mean values of several determinations shown. Similar results were obtained in aorta from control and diabetic rats.

Western blot and densitometry. Aorta and caudal artery membranes from control and diabetic rats were blotted with AS/7 (which recognizes G_i1,2), EC/2 (which recognizes G_i3/0) and QL (which recognizes G_q/11). Each antibody produced one major band (fig. 4). Aliquots of the same human platelet membrane sample were used as an internal standard with this series of blots. A single band was detected in the platelet sample at the same molecular weight as the band seen with each of the control and diabetic aorta and caudal artery samples (fig. 4). The standard was run for each antisera on each blot to correct the control and diabetic artery samples for any blot-to-blot variations. After densitometric analysis, no significant differences in the levels of immunoreactive protein were detected between control and diabetic rats in either aorta or caudal artery membranes (fig. 5).

View larger version (65K): [in this window] [in a new window]   Fig. 4.   Representative Western blots of aorta and caudal artery membranes from control (C) and diabetic (D) rats, with adjacent human platelet (P) standards using the three antisera AS/7, EC/2 and QL.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Densitometric analysis of Western blots of aorta (n = 5 or 6) and caudal (n = 5 or 6) artery membranes from control (unfilled bars) and diabetic (filled bars) rats. Results are expressed in arbitrary units corrected for membrane protein and the relative levels of the immunoreactive protein in the platelet standard.

	Discussion

Abstract Introduction Methods Results Discussion References

The present study has found a significant decrease in the number of alpha-1 adrenoceptors in caudal artery membranes from 12- to 14-week STZ-diabetic rats compared with age-matched control caudal artery membranes, whereas no difference in alpha-1 adrenoceptor number was detected between control and diabetic aorta membranes. No change in affinity of the alpha-1 adrenoceptor for antagonist or in G protein levels was detected between control and diabetic rat arteries. However, the coupling of G proteins to the alpha-1 adrenoceptor was found to be impaired in both aorta and caudal arteries from diabetic rats. Thus, not only do changes in the number and coupling of the alpha-1 adrenoceptor or levels of G proteins not explain the enhanced contractile responses in diabetic arteries, the alpha-1 adrenoceptor changes that we measured would tend to counteract the enhancement. Instead, an increase in the activity of the G proteins or PLC- coupled to the alpha-1 adrenoceptor may be mediating the enhanced responsiveness elicited by the alpha-1 adrenoceptor in diabetic arteries.

Changes in alpha-1 adrenoceptor number and affinity. The estimated K_d of the alpha-1 adrenoceptor for the specific antagonist prazosin in both aorta and caudal artery membranes prepared from control and diabetic rats falls within the range of previously reported values (0.10-0.65 nM) in various arteries (Agrawal and Daniel, 1985; Cheung and Triggle, 1988; Jagadeesh et al., 1991). Thus, we have measured a single, saturable binding site that corresponds to the alpha-1 adrenoceptor in its affinity for prazosin in control and diabetic aorta and caudal artery membranes. However, the B_max values obtained in the present set of experiments, particularly in the caudal artery, are 4 to 10 times higher than the values reported by these other investigators. This could be partly due to differences in methodology between our study and those previously reported. In addition, a previous study in bovine aorta (Jagadeesh et al., 1991) demonstrated that the number of alpha-1 adrenoceptors increased with the age of the animal. The rats used in the present study were much larger (450-600 g for control) than those used in other alpha-1 adrenoceptor binding studies (200-300 g; Agrawal and Daniel, 1985; Cheung and Triggle, 1988), making an age-induced increase in alpha-1 adrenoceptor number a likely contributor to our relatively high B_max values.

Effect of guanine nucleotides on affinity of the alpha-1 adrenoceptor. Under experimental conditions in the complete absence of GTP, a high-affinity complex of receptor and heterotrimeric G protein can be detected in agonist radioligand binding experiments. In the presence of agonist and absence of GTP analogs, only a fraction of receptors will be detected in the high-affinity state; most are in the low-affinity state (DeLean et al., 1980). When GTP analogs are present, only a single low-affinity agonist binding site is detected on the receptor. However, it has been speculated that the low-affinity state of the receptor may not preclude activation of G proteins; it may only require higher concentrations of agonist to elicit activation (Jagadeesh and Deth, 1987).

q/11

Effect of diabetes on G protein levels in arteries. A considerable amount of evidence has accumulated to show that levels of certain G proteins, namely G_i, decrease in hepatocytes of human diabetics and STZ-diabetic rats, whereas the levels of G_s increase slightly (Bushfield et al., 1990; Caro et al., 1994). In adipocytes, no change in the level but instead an impaired functioning of G_i was found in STZ-diabetic rats (Green and Johnson, 1991). However, an increase in the level and functioning of G_i was reported in adipocytes from a genetic model of diabetes (Strassheim et al., 1991). Platelets from diabetic humans had severely diminished levels of G_i compared with nondiabetic human platelets (Livingstone et al., 1991), whereas diabetic retina showed impaired functioning or decreased levels of G_i (Hadjiconstantinou et al., 1988). These diabetes-induced changes in G_i and G_s may relate to the recent discovery that G_i2 is linked to signaling of the insulin receptor, and a complete knockout of the G_i2 gene produces a metabolic state that resembles type II diabetes mellitus in mice (Moxham and Malbon, 1996). No study has examined the effect of diabetes on G proteins in vascular tissue.

i2,3

q/11