a-hdrenergic supersensitivity and decreased number of a-adrenoceptors in heart from acute diabetic rats

MIRIAM WALD, ENRI S. BBRBA, AND LEONOR STERIN-BBIPDA' Centrol de Estudios Farmacol6gicos y de Brincipios Naturales (CEFAPRIN), Consejo Nacisnal de Investigacisnes CientSficws

y Tbcnicas de la Repdb&ic~ Argentina (CONICET)'), Serrano 665/69, (4414) Buenos Aires, Argentina

Received November 16, 1983

WALD, M., ENRI, S . B . , and STEWIN-BORDA, L. 1988. a-Adrenergic supersensitivity and decreased number of a-adrenoceptors in hem from acute diabetic rats. Can. J. Physiol. Phmacol . 66: 1 154- 1 160.

The inotropic effect of methoximine, as well as the a-adrenoceptor population, were measured in cardiac tissue from nomal and short-term 43 days) diabetic rats. Mehoxamine increased the tension of both nomal and diabetic ventricles, but in diabetic ones, the dose-response curve to methoxamine was shifted to the left and the efficacy of the a-agonist was enhanced. This phenomenon was accompanied by an increase in receptor affinity, while the number of a-adrenoceptor sites decreased. Inhibitors of al-ad~noceptors blocked, in a competitive manner, the positive inotropic effect of methoxamine in both types of ventricles. Inhibition of phospholipase C blocked the ventricular response to the methoxamine in nondiabetic as well as in diabetic hems. Synthetic diacylglyceride (DAG) potentiated the inotropic action of the a-agonist in normal ventricles and increased the affinity with a decreased number of a-adrenoceptor sites in normal ventricles, producing values of Kd and B,,, similar to those of the acute diabetic heart. Inhibitors of protein kinase C partially reduced the supersensitivity to a-agonists in diabetic ventricles and prevented the stimulatory action of DAG upon the positive inotropic effect of methoxamine in normal ventricles. These results suggest that a-adrenergic inotropic stimulation is secondary to receptor-mediated hydrolysis of phosphoinositides, generating some oxidative metabolites (DAG) which, in t m , may be responsible for the inotropic effect. In the acute diabetic state, the supersensitivity to a-agonist could be due to high activity of phospkolipase C (with an increase in DAG production) which induces alteration in the membrane a-akenergic receptors.

WALD, M., EN^, S. B., et S T E R I N - B B ~ A , L. 1988. a-Adrenergic supersensitivity and decreased number of a-adrenoceptors in heart from acute diabetic rats. Can. 9. Physiol. Phmacol . 66 : I 154- 1 160.

On a examin6 l'effet inotrope de la mCthoxhne ainsi que la population des a-adknwepteurs dans le tissu cudiaque de rats nomaux et rendus diaMtiques depuis peu (3 jours). La m6thoxamine a augment6 la tension des ventricules nomaux et diabktiques, mais chez les BjiabCtiques, la courbe dose-rCpnse h la m6thoxamine a CtC d6calCe vers la gauche et l'efficacitk Be I'a-agoniste a kt6 accrue. Ce ph6nodne a Ct6 accompagn6 d'une augmentation de H'affinitC du rkcegteur, alors que le nombre Be sites d'a-adrCnocepteurs a diHninuC. Les inhibiteurs d'al-adrknwepteurs ont bloquC de mani$re compititive l'effet inowope psi t i f de la m6thoxarnine dans les deux types de ventricules. L'inhibition de la phospholipase C a bloqu6 la rCponse ventriculaire ii la mCthoxamine dans les c o e m non diabktiques et diaMtiques. Le diacylglycCride (DAG) synthetique a potentialis6 l'action inotrope de l'a-agoniste dans les ventricules nomaux et a augment6 B'affinitC avec un nombre ruu i t de sites d'a-adr6noceptet.u-s dans les ventricules nomaux, prduisant des valeurs de Kd et de B,, shilaires B celIes du coeur diaMtique aigu. Les inhibiteurs de prot6ine kinase C ont rCduit pmiellement I'hypenensibilitC aux a-agonistes dans les ventricules diawtiques et ont prkvenu l'action stimulatrice du DAG sur l'effet inotrope positif de la rnkthoxamine dams les ventricules nomaux. Ces rksulhts suggkrent qu'une stimulation inotrope a-adrknergique est secomdaire a l'hydrolyse des phosphoinositides mtdiCe par les rkcepteurs, produisant quelques mktabolites oxydatifs (DAG) qui, en retour, pourraient Ctre responsables de l'effet inotrope. A 1'Ctat diabktique aigu, 19hyper~en~ibilit6 B I'a-agoniste poumait Ctre due h une grande activitk de phospholipase C (avec une production accrue de DAG) qui provoque 19alteration des rkcepteurs a-adknergiques de la membrane.

[Traduit par la revue]

Diabetes has been associated with an autonomic neuropathy. The sympathetic nervous system appears to be activated in the early stages of diabetes, because plasma catecholamines have been reported to be elevated (Christensen 1974; Berkowitz et al. 1980; Paulson et al. 1980). Plasma catecholamine levels have also been found to be unaffected (Cryer et al. 1978) or depressed (Christensen 1972; Hepeldtke et d. 1982) during diabetes. Likewise, diabetic cardiac catecholamine concentrations have been described to be decreased (Neubauer and Christensen 1976) or increased (Paulsow et al. 1980; Fushimi et al. 1982.)

It has been detected that the responsiveness of the diabetic myocardium to adrenoceptor agonists is altered (Foy and Lucas 1976, 1978). Changes in sensitivity of a-adrenoceptor agonists have been observed in tissues isolated from diabetic rats.

' ~u tho r to whom correspondence should be sent at the following address: CEFAPRIN, Serrano 665, (11414) Buenos Akes, Argentina.

However, there are discrepancies in the results reported about these changes. Warnanandham et al. (1 984.) reported not only increased sensitivity of tail artery strips but also a decreased response to agonists in thoracic aorta isolated fmm diabetic rats. Meyliger et al. (1982) described a decrease in a-adrenoceptsr density in the myocardium from chronic diabetic rats, accompa- nied with a depressed contractile responsiveness to methoxam- ine. This observation contrasts with preliminary reports in which a supersensitivity was found to the inotropic action of methoxamine and phenylephine in diabetic rat atria (Canga and Sterin-Borda 1986; Jackson et al. 1986) and in lamb hearts (Downing et d. 1983). A supersensitivity to the a-adrenergic effect of norepinephrine in vasa deferentia has also been described (Peredo et al. 1984).

In a recent study (Canga and Sterin-Borda 1986) we suggested that the supersensitivity to methoxamine observed in atria isolated from acute diabetic rats could be mediated by a mechanism that couples activation of cardiac a-adrenoceptors

Wnted in Canada / Imprid am Cmda

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WALD ET Ak.

to changes in the metabolism of arachidonic acid towards an pt.Qduction of tkromboxme (TX)A2 and alipoxygenase(s)

derived product. Futhermore, we have established that in the diabetic state the metabolism sf arachidonic acid is shifted toward TXB2 formation, while in normal atria it is directed toward PG12 production (Canga et d. 1984).

In this paper it will be shown that supersensitivity to an a-adrenergic agonist in the ventricular myocardium from acutely diabetic rats is accompanied by a reduced number of a-adrenergic receptor binding sites and an increase in their affinity constants. The mechanism appears to involve the activation of polyphosphoinositide tunrover that could be enhanced by the diabetic condition.

Material and methods Anim&s

Experimental short-term diabetes was induced as previously report- ed (Canga and Sterin-Borda 1986; Canga et al. 1984) in Wism male rats (200-250 g) given a single i.p. dose of streptozotocin (STZ; 85 mg . kg-' body weight, Sigma Chemical Co.). STZ was dissolved in citrate buffer (pH 4.8) prior to the injection. A colorimetric enzymatic methad (glycemia enzymatic) for ""true glucose" determination in blood was used. Animals with plasma glucose above 300 mg . d ~ - ' and with glycosuria were considered diabetics. The rats were killed 72 h after ST2 injection.

Preparation ofpurijied membranes and binding assay Diabetic and nomal cardiac membranes for identification of

a-adrenweptors were prepared essentially as described by Limas and Limas (1978) and Borda et al. (1984). Briefly, venticular tissue from eight rats was mixed in 4 vol. of cold buffer containing 0.25 m sucrose, 5 rraEvI Tris-HC1 (pH 7.4), and 1 mM MgC12, and was homogenized with a Polytron PT-20 at a setting of 3-5 for 10- 15 s, three times. The homogenate was centrifuged at 1080g for 10 min. The supernatant was centrifuged at 1 2 OOOg for 10 min and then at 30 OOOg for 120 min. The gellet was resuspended in 2-5 mL of 50 mM Tris-HC1 (pH 7.4) and 10 mkul MgC12. The membrane suspension thus contained 2-3 mg/mL of protein. Relative activity of 5'-nucleotidase indicates the degree of purity of the microsomal fraction (Table 1); enzyme activities werc similar in noranal and diabetic conditions.

For [ '~]~razosin binding, 100 FL of membrane suspension and different concentrations of (-)[3~]flprazosin (New England Nuclear Company, specific activity 82 Ci /mol ) (1 Ci = 37 GBq) were incubated with shaking for 20 min at 25°C in a total volume of 150 pL of 50 hnIW Tris-HCl (pH 7.4) and 10 mM MgC12. At the end of the incubation period 150 pL was placed into 4 mh, of buffer and immediately filtered through GF/c glass fibre filters. The filters were washed with 8 d of buffer, dried, added to 10 d of Triton-toluene based scintillation fluid, and counted. Nonspecific binding was determined by filtering the membranes incubated in the presence of 10-'ha (+)phentolamine; it did not exced 10% of the specific binding.

Specific binding rcsults are expressed as femtomoles of (-)- ['H]prazosin bound per milligram of protein.

Isolated ventricle preparations to measure contractile activity The animals were killed by decapitation with a guillotine. The entire

heart was quickly removed and placed in Petri dishes filled with a modified Krebs-Ringer-bicarbonate (KRB) solution of the following composition (mM): Na9, 145; K9, 6.02; Ca9, 1.22; Mg9, 1.33; C1-, 126; HC03-, 25.3; SO^^-, 1.33; P042-, 1.20; and glucose, 5.5 (Sterin-Borda et al. 1983). fight ventricle strips were cut, attached to a glass holder, and immersed in a tissue chamber filled with 20 mL of KRB solution, gassed with 95% 02--5% C02, and kept at 30°C and pH 7.4 throughout the experiments. One end of the preparation was anchored to the glass holder and the other was connected to a force transducer (Stratham UC-3 Gold Cell) coupled to an ink writing oscdlograph (SAN Em 180). A c o t ~ t m t resting tension of 1 g was

TABLE 1. 5'-Nucleotidase activity

Conditions Homogenate Microsoms

Normal 0.15 + 0.04 1.59 & 0.05 Diabetic 0.14 2 0.05 1.52 + 0.06

NOTE: Vdues are expressed as micxornoles of Pi p a milli- g m of protein in 10 min. Vdues represent the mems 2 SEM of six experiments in each group.

applied to the strips by means of a micrometric device. Right ventricle strips were electrically excited with field stimulation using square wave pulses of 1-ms duration and slightly suprathreshold intensity (10%) at a frequency of 1 Hz delivered from a conventional stimulator.

The mechanical activity was analyzed in terms of maximum rate of isometric force (dF/dt) developed above the externally applied resting tension. Tissues were allowed to equilibrate for BO min. Control values (100%) refer to dF/dt before drug administration. Absolute values of dF/dt at the end of equilibration period (60 min) were 16.5 + 1.6 g/s for nomal and 15.7 & 1.2 g/s for diabetic ventricles.

Cumulative dose-response curves for methoxamine were made according to the method described by Van Rossum (1963). Single doses were administered in volumes of 0.01-0.025 mL of KRB solution. The total volumes added to the bath never exceeded 0.1 mL. The time interval between doses was that required for each to produce a maximal effect (10 min).

Drugs Freshly prepared solutions of the following drugs were used:

methoxamine (Met) from Burroughs Wellcome; L-propranolol from Ayerst; and phentolamine, prazosin, 2-nitro-4-carboxyphenyl-M,M- diphenylcarbamate (NCDC) , 1 -01eoyl-2-acetyl- sn-glycerol (BAG), norepinephrine, isoproterenol, 1 -(5-isoquinoliny1sulfonyl)-2-methyl- pigemhe dhychxhlaride (W-7), and N-(2-aminoethy1)-5-iquinoline sulfonamide dihydrochloride (H-9), (all from Sigma Chemical Co.). All h g s were diluted in the organ bath to achieve the final concentration shown in Tables 3 and 4 and Fig. 2. Ventricle strips were incubated for 30 min with a-adrenoceptor antagonists and (or) inhibitors of phospholipase C (PLC) or DAG before adding increasing amounts of Met.

Statistics The Student's t-test for unpaired values was used to determine the

levels of significance. Differences between means were considered significant when p 5 0.05.

Results Binding assay

[3~]~razos in binding to cardiac membrane was a saturable process, with half-maximal saturation occurring in normal ventricles at 0.36 k 0.07 1 nM and in diabetic ventricles at 0.20 k 0.038 nM (Fig. la). A Scatchard plot of specific ['~lprazosin binding tQ cardiac membranes is shown in Fig. 1 b. The intercept with the abscissa indicates the binding sites (B,,), whereas the negative reciprocal of the slope provides an estimate of the equilibrium dissociation constant (Kd) for the interaction of ['~lprazosin with the binding sites. At saturating [ '~l~razosin concentrations (0.75 and 1.50 nM in diabetic and normal tissue respectively), specific biding accounted for 90% of the total, while at lower concentrations it accounted for 94%. As shown in Fig. l a , more ['Hlprazosin was specifically bound to normal membrane preparations than to diabetic membranes at six concentrations of the radioligmd employed.

Cumulative Scatchard analysis of the data demonstrated a statistically significant decrease in the maximum number of binding sites with an increase in the affinity constant in the

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CAN. J . PHYSIOE. PHARMACOE. VOL. 66, 1988

FIG. 1. (a) Specific binding of ['~]~razosin to rat cardiac mem- branes in control ( a ) and diabetic ( 0 ) rats. Specific binding is defined as total binding minus nonspecific binding as measured in the presence and absence of 10-~h% phentolmine (expressed as fmol/mg protein). (b) Scatchard plot of saturation binding data from (a). The ratio of bound [3~]prazosin (fmol/mg rotein) to free [3~]prazosin (m) is P plotted as a function of the [ H]praz~sin bound per milligram of protein. The intercept with the abscissa is the number of binding sites (B,,) and the negative reciprocal of the slope is the dissociation constant (I&). Results are the means + SEM of eight experiments in each group assayed in triplicate.

TABLE 2. Binding of [3~]prazosin to cardiac membranes from control and diabetic rats: effect of DAG

Diabetic 88.42*+ 6.94 8.224* k8.0$0 Diabetic + DAG 96.02*& 7.05 8.159*k0.018 Normal l69.48+ 13.44 0.415k0.062 N o m d f DAG 95.03*k 7.03 0. 185*+0.020

NOTE: Membranes ( lW pL) were incubated for 20 mh with different concentxations sf ['~]pmzosh in the presence or absence of BAG (5 X M). Vdues represent the anems 2 SEM d eight experiments in each p u p , assayed in triplicate.

*Significantly different from n o m d control values ( p < 0.001). Statistics were deter- mined using a simple computerprogram for Scatchard plot analysis (Sagripanti et al. 1984).

cardiac membranes of diabetic rats (Fig. 1 b and Table 2). These differences were also observed with methoxamine competition binding assay (nomal, Ki = 41.0 If '7.2 pM; diabetic, Ki = 10.5 k 3.1 pM; n = 4).

Contractile assay To detemine if a-adrenergic receptor alteration observed

during diabetes may have influenced the mechanical response to a-agonist, the effect of Met on dF/dt of nomal and diabetic ventricles was examined. Figure 2 shows the effect of increasing concentrations of Met on the contractility of ventricles from nomd and diabetic rats. It can be seen that Met induced a coneen~tion-dependent increase in dF/dt in both groups of ventricles. Both the efficacy (Emax) and potency (Kd) were greater in ventricles from diabetic rats (Ern, = 197 If 14g/s; Kd = 0.85 X M) than those from normal control rats (Em, = 92 2 $1 g/s; = 2-04 x MI.

To assess the role of a-adrenoceptors in the contractile effect sf Met, ventricles were incubated with a-adrenoceptor antago- nists such as prazosin md phentolmine. The result$ shown in

Met hoxarni ne $ - Iog M 1

FIG. 2. Cumulative dose-response curves to methoxamine in ven- tricles isolated from ,normal ( 0 ) and diabetic ( a ) rats. dF/db is expressed as percent change vs. initial controls. Each point represents the mean; vertical lines are SEM. Numbers in paentheses represent the number of experiments. p<0.001 between m m a l and diabetic ventricles.

Table 3 indicate that in the presence of these agents, the positive inotropic action of Met was antagonized in a competitive manner both in n o m d ventricles and in those from diabetic rats. To demonstrate that the effects sf Met were mediated via the a a -adrenoceptors, additional experiments were conducted in the presence of propranolol M). The lack of action of the B-adrenoceptor bbcker is seen in Table 3. The dF/dt values developed after the addition of the a-antagonists in both normal and diabetic ventricles had similar magnitudes (Table 4).

To detemine the nature of the mechanism triggering the supersensitivity to Met in diabetic ventricles, the action of an inhibitor of PLC was explored. As can be seen in Fig. 3, NCDC reduced in a concentration-dependent manner the inotropic positive effect of Met in diabetic ventricles, reaching values similar to those observed in n o m d ventricles. NCDC also inhibited the action of Met in n o m d ventricles.

To elucidate which product from polyphosphoinositide tum- over could be involved in this effect, ventricles were incubated with DAG for 45 min. As can be seen in Fig. 3, in the presence of DAG the stimulant effect of Met was significantly increased in normal ventricles, reaching values similar to those observed in the diabetic conditions. At this concentration, DAG did not modify the dose-response curve of Met in diabetic ventricles, but at higher concentrations of DAG (5 X M) the stirnula- tory action of Met M) was enhanced by about 50% in the diabetic condition. NCBC and DAG did not modify the dF/dt of normal and

diabetic ventricles prior to the addition of the al-adrenergic agonist (Table 4). It is important to note that NCDC or DAG did not modify the inotropic action of norepinephrine and isoproter- enol, demonstrating the specificity of these agents for the al-adrenergic stimulation (data not shown). These findings rule out a nonspecific effect of the drugs on contractility. To

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TABLE 3. Influence of a- and p-adrenoceptor antagonists on the postitive in~tropic effect of methoxamine in normal and diabetic atria

Norrnal ' Diabetic

h g s ~ m ( g / s ) Kd( x lo-") Em,,(g/s) Kd( x 10-6w

Methoxamnine 9428 2.04 197+ 14 0.85 Phentolamine (10-%) 89k7 7.50* 1892 10 9.20* Prazosin (lo-%) 9026 6.90' 1922 11 8.70* hpranolol( 1 o-'M) 9628 2.08 195213 0.87

NOTE: Values are means + S I N of seven experiments in each group. *Statistically different from control values (g<0.0001).

FIG. 3. Contractile effect of methoxmine (0-0) in diabetic (a) and normal (b) ventricles. Influence of NCDC at 1 X ~ o - ~ M (9.. ..) and 2 X ~ o - ~ M (0.. .O) , and DAG at 5 x ~ o - ~ M (0-0). Each point represents the mean; vertical lines ape SEM. Numbers in parentheses represent the number of experiments. p < 0.001 between methoxarnine alone vs. NCDC in (a) and (6 ) . p < 0.001 between methoxarnine alone vs. DAG plus methoxmine in (b).

TABLE 4. Effect of different drugs on the dF/dt of rat isolated ventricle prevented the stimulatory action of BAG on the positive inotropic effect of Met in normal ventricles. Neither H-7 nor H-9

Normal ventricle Diabetic ventricle modified the inotropic action of Met on normal ventricles nor the Dmgs dF/dt (g/s) dF/dt (g/s) basal dF/dt of normal or diabetic ventricles (Table 4).

The ability of DAG to modify [3~]prazosin binding in both None 16.55~ 1.6 (7) 15s7+ e 2 (7) normal and diabetic cardiac membranes was also tested. It can Razosin (lo-") 15.82 1.4 (5) 160422.1 (5) be observed in Table 2 that in the presence of DAG the B,,, Phentolamine M) 14.92 1.3 (5) NCDC (5 x los%) 16.22 1.9 (6)

15* .9 decreases and the affinity constants increase in cardiac mem- 14.82 1.5 (6) DAG (5 X M) 15.622.0 (6) 1 1 5 ( ) branes from normal rats, reaching values similar to those H-7 M) 15.92 2.7 (6) 15 .72 .7 (6) observed in cardiac membranes from diabetic rats.

NOTE: Values are means A SEM. Initial control values (no additions) were recorded - after an equilibration period (HI min following set-up of the preparation). Experimental values were morded 30 min after administration of each drug (see Materials and methods). Values in parentheses are the number of preparations tested.

determine whether the protein kinase C (PKC) is implicated in the supersensitivity to Met observed in diabetic ventricles, the action of H-7 and H-9 inhibitors of the PKC activity (Hidaka et al. 1984; Inagaki et al. 1985) was studied. As shown in Fig. 4 both H-7 and H-9 partially reduced the inotropic effect of Met in diabetic ventricles. In addition, the inhibition of PKC activity

Discussion The present results demonstrate that acute diabetic rat

ventricular muscle exhibits an increased ability to respond to methoxamine. This phenomenon, which appears to be second- ary to receptor-mediated hydrolysis of phosphoinositides, is accompanied by an increase in the receptor affinity, while the number of a-adrenoceptor sites decreases.

The pharmacological analysis of both a-agonist and antago- nist tends to support the idea that the al-adrenoceptors are the most important mediators of the positive inotropic response to methsxamine in both diabetic and normal ventricles. The

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CAN. B. PHYSIOL. PHARMACOL. VOL. 66, 1988

1 0 9 8 7 6 5 4 3 2 1 0 9 8 7 6 5 4 3 2 Met hcsxamine I - log M I

FIG. 4. Contractile effect of rnethoxamine (a-0) in diabetic ventricles (a) and in normal ventricles exposed to 5 X I O - ~ M DAG (b ) . Influence of 1 x IO-~M H-7 (@.. . @) and 1 X 1W5hl H-9 ( 0 . . . 0 ) . Other details as in Fig. 3. Ventricles were incubated 20 min with H-7 and H-9 prior to the addition of methoxamine (a) or methoxamine plus DAG ( l a ) .

positive inotropism elicited by stimulation of post-junctional a-adrenoceptors has been described in isolated myocardium of various mammalian species (Wagner and Brodde 1978; Schii- man et al. 19781, including isolated rat atria and ventricles electrically paced (Wenzel and Su 1966; Shibata et al. 1980). However, little is hown abut myocardial mctivity to a-agonists during an acute diabetic state. At a time when the isolated work- ing heart has dF/dt values similar to those of nomal control (Table 41, were found that ventricles from diabetic rats were supersensitive (decreased Kd) and hyperresponsive (increased Em,) to selective a-adrenoceptor stimulation by methoxamine.

Therefore, the affinity of al-adrenoceptor for methoxamine in myocardium from diabetic rats is increased. This observation is consistent with data from receptor binding studies (this paper) in which an increase in the affinity was observed (decreased Kd) but was accompanied by a decrease in a-receptor density (B,,,) during acute diabetic conditions. Although it is hazardous to correlate antagonist binding data with functional agonist activi- ty, it is possible that the increase in sensitivity in light of a decrease in a-adrenoceptor density is linked to an increase in the receptor affinity. Data obtained with the agonist competition binding assay appear to confirm this. The decrease in receptor density could, on tfie other hand, be a compensatory measure to maintain normal cellular response to al-agonist.

A dissociation between the mechanical effect of the agonists and the number of receptor sites has been described in short-term diabetic rats. Carrier et al. (1984) reported a post-functional muscainic supersensitivity to the negative chonotropic effects of cholinergic agonists accompanied by a decrease in the atrid muscainic receptors in the diabetic state. Moreover, hyperresponsiveness to the contractile effect of a-agonists was also found in isolated left atria from chronic (3 months) diabetic rats (Jackson et al. 1986) and in atria from acute diabetic rats (Canga et al. 1984). Downing et al. (1983) observed an increase in the inotropic (dF/dt) response to methoxmine in diabetic lamb hearts as early as 2 days after injection with alloxan. On the contrary, during chronic diabetic

conditions a reduction in the inotropic response of ventricular muscle to methoxamine was described, coinciding with a decrease in a-adrenoceptor density of the heart, without changes in receptor affinity (Heyliger et al. 1982; Williams et al. 1983; Eatifpour and McNeill 1984).

The increase in the intrinsic activity of the a-agonist observed in this paper could be due to an alteration in the utilization of calcium by the myocardium, after receptor occupancy-mediated hydrolysis of phosphoinositides, as described for some of the physiological and metabolic effects of a*-adrenergic stimula- tion on rat myocardium (Brown et al. 1985).

The observation presented here that the positive inotropic effect of methoxamine was abolished by NCDC at a concentra- tion hown to inhibit phospholipase C (Walenga et al. 1980) supports the notion that in isolated rat ventricles, the response is initiated by an increase in the hydrolysis of the phosphoinosi- tides. The fact that the hyperreactivity of diabetic heart to the a-agonist was also abolished by NCDC could support the hypothesis that in the acute diabetic heart the activity of PLC is greater than in the normal condition. To determine which PLC end-products participate in this event, both groups of ventricles were exposed to synthetic DAG before testing their respnsive- ness to mehoxamine. In the presence of DAG, normal ventricles showed a hyperreactivity to the a-agonist similar to diabetic ones.

Moreover, DAG increased the affinity and decreased the a-recepbr density in nomal conditions producing values of Kd and B,,, similar to those observed during the acute diabetic state. Thus, BAG may affect the behaviour of the nomal heart in that it responds like a diabetic hem. Although NCDC is not a specific inhibitor of PLC, the fact that NCDC and DAG did not modify either the inotropic action of other adrenergic agsnists (norepinephrine and isoproterenol) or the basal dF/dt rules out a nmspific interaction of the h g for a-adrenergic stimulation.

One of the best hown effects of BAG is the activation of PKC. It is logical to suppose that these results are secondary to the activation of the enzyme by DAG. The fact that the

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inhibition of PKC reduced the inotropic effect of methoxmine in the diabetic condition and attenuated the stimulatory action of DAG on the methoxamine effect in normal ventricles supports this notion. However, because the inhibition of PKC only partially blocked the inompic action of Met, it is not possible to rule out the participation of inositol 1,4,5-eksphosphate as a messenger in the supersensitivity to a-agonists in the diabetic state. The ability of DAG to modify [3~]prazosin binding in normal heart, reaching values similar to those observed in the diabetic condition, is in accordance with the concept that one of the magor functions of protein kinase C appears to be intimately related to a negative feedback control on the a,-adrenoceptor, termed down-regulation (Nishizuka 1986). For methoxmine supersensitivity an additional mechanism could be implicated, such as an alteration in the utilization of calcium and (or) in the production of a series of biologically active arachidonic acid metabolities. This speculation is in accordance with our previous report in which we showed that the hypersensitivity to methoxamine observed in the diabetic state is mediated by some eicosanoids (Canga 1986). Furthermore, hypersensitivity to calcium associated with an increased sarcolernmal Ca2+ AFPase activity in acute diabetic rat heart has also been ob- served (Borda et al. 1987).

M i l e the evidence provided in this study is indirect, it is consistent with the hypothesis that a-adrenergic stimulation of the heart could be associated with the receptor-mediated hydro- lysis phosphoinositides. In the acute diabetic condition, the increase in the intrinsic activity of a-adrenergic agonists could be due to increased activity of PLC, which triggers cascade reactions inducing alterations in the membrane a-adrenergic receptors.

Acknowledgements This work has been supported by grant 3905081/02-85 from

CQNICEF, Argentina. We also extend thanks to Mrs. Elvita V m u c c h i and Mrs. Beatriz Costigliolo for technical assistance and typing of the manuscript, respectively.

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