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Longitudinal muscles from the ileum of rats rendered diabetic for 10 to 12 weeks by injection of streptozotocin (STZ) developed greater contractile force in response to muscarinic agonists, (acetylcholine, carbamylcholine and bethanechol) than muscles from age-matched control rats. There was no change, however, in the sensitivity of longitudinal smooth muscles to muscarinic agonists as reflected by the EC50 values for stimulation of muscle contraction. This increased responsiveness of the muscles was accompanied by a 32% reduction in the density of muscarinic receptors (381 +/- 93 vs. 560 +/- 74 fmol/mg of membrane protein) in muscles from STZ-diabetic rats. There was no change in agonist or antagonist binding affinities in muscles from diabetic rats, and there was no alteration in the distribution of receptors between the states characterized by high and low affinity agonist binding. There was also no change in acetylcholinesterase activity in muscle membranes isolated from STZ-diabetic rats. The origin of the increased responsiveness of ileal smooth muscles in this model of diabetes is not clear, but may involve changes in the muscarinic signal transduction pathway beyond the receptor level, or in the contractile apparatus itself.

Previous results from our laboratory (White and Carrier, Enhanced Vascular Alpha-Adrenergic Neuroeffector System in Diabetes: Importance of Calcium. Am. J. Physiol. 255: H1036-1042, 1988) demonstrated that mesenteric arteries from streptozotocin (STZ)-diabetic rats exhibit an enhanced responsiveness to alpha adrenergic agonists. The present study demonstrates that this enhanced responsiveness is dependent upon the presence of extracellular calcium. Arteries from STZ-diabetic (10-12 weeks) rats developed greater contractile force in response to norepinephrine or KCl. Development of these effects was prevented by daily insulin treatment, indicating these alterations are related to the diabetic state. Similarly, the contractile response to extracellular calcium in the presence of norepinephrine (3 x 10(-6) M) or KCl (60 mM) was greater in arteries from STZ-diabetic animals. BAY K 8644, a calcium channel agonist, induced greater contraction in arteries from STZ-diabetic animals, as did activation of protein kinase C by phorbol dibutyrate. In contrast, contraction induced by release of calcium from intracellular sources (alpha-1 adrenoceptor-mediated or caffeine-induced) was unaltered by diabetes. These findings indicate that enhanced vascular contraction in STZ-diabetes is of a nonspecific nature, i.e., the contractile response to any agent which induces extracellular calcium-dependent contraction should be enhanced in diabetes. We propose that STZ-diabetes enhances the activity and/or number of calcium ion channels in vascular smooth muscle.

Autonomic neuropathy is a major complication of chronic diabetes and is responsible for disturbances in the cardiovascular system and other organs. Early cardiac disturbances have been attributed to defective vagal control of the heart. The heart rates of rats with chemically-induced diabetes are depressed and an increase in blood pressure produces a greater reflex bradycardia in diabetic rats. Tomlinson and Yusof found that isolated, stimulated left atria from rats made diabetic with alloxan are supersensitive to the negative inotropic influence of acetylcholine. On the other hand, Rao, et al. found that perfused working-heart preparations from streptozotocin- and alloxan-diabetic rats have a reduced sensitivity to carbamylcholine. In the present study, we measured chronotropic responses to cholinergic agonists of isolated, spontaneously-beating atria, as well as muscarinic receptor populations, in cardiac tissue from short-term (8 to 9 weeks) diabetic and age-matched control rats.

Hypertension, atherosclerosis, arteriosclerosis, and cardiomyopathy account for 80% of all diabetic deaths. A paucity of data exists concerning reactivity of the diabetic vasculature to vasopressor substances. The purpose of this investigation was to determine the influence of short-term (4-5 weeks) diabetes on the responsiveness of the cardiovascular system to vasopressor substances in the intact conscious rat. Diabetes was chemically induced with streptozotocin (65 mg/kg i.v.) in 42-43-days-old, nonfasted, male Sprague-Dawley--derived rats. Direct blood pressure and heart rate were recorded, and responses to the vasopressor agonists norepinephrine and angiotensin II were obtained. Short-term diabetic rats were hypotensive and had lower heart rates when compared with control rats. Blood pressure responses to norepinephrine and angiotensin II were depressed in the short-term diabetic rat; however, the baroreceptor reflexes in these rats were more sensitive to increases in blood pressure. Therefore, it appears that there is some type of nonspecific alteration in the responsiveness of the cardiovascular system to the vasopressor agonists in the short-term diabetic rat.

1 In the present investigation, noradrenaline-induced vascular contractions in isolated rat mesenteric arteries were studied in short and long-term experimental diabetic rats. The diabetic state was induced with streptozotocin (65 mg/kg, i.v.). 2 Long-term diabetic rats were hypertensive when compared to age-matched control rats. In contrast, short-term diabetic rats appeared to be hypotensive when compared to age-matched control rats. 3 Long-term diabetic mesenteric arteries were supersensitive to noradrenaline and developed more contractile force in response to noradrenaline when compared to age-matched control mesenteric arteries. beta-adrenoreceptor antagonism had no effect on the sensitivity of the mesenteric arteries (diabetic and control) for noradrenaline, however, there was an increase in the contractile force developed by these tissues in response to noradrenaline during beta-adrenoreceptor antagonism. 4 alpha-Adrenoreceptor antagonism produced a decrease in the sensitivity of both long-term diabetic and corresponding age-matched control mesenteric arteries. alpha-Adrenoreceptor antagonism had no effect on contractile force developed by long-term mesenteric arteries (diabetic and control) in response to noradrenaline. 5 The present data indicate that long-term experimental diabetes produces a supersensitivity of rat mesenteric arteries to noradrenaline. This phenomenon is dependent upon the duration of the diabetic state. Supersensitivity of the mesenteric arteries to noradrenaline may indicate an increased arterial vascular resistance which may contribute to the observed hypertension.

Contractile responses of aortic ring preparations from control and diabetic rats to nonselective (noradrenaline) and selective alpha 1-(phenylephrine) and alpha 2-(clonidine) adrenoreceptor agonists were examined to determine the contribution of these receptor subtypes to the response. Aortae from diabetic rats were more responsive to noradrenaline compared to age-matched control rats. There was no difference between contractile responses of aortae from control and diabetic rats to phenylephrine, whereas clonidine-induced contractions were enhanced in aortae from diabetic rats. Dependence of clonidine-induced contractions on extracellular calcium availability was determined. Each of the methods used indicated increased dependency of aortae from diabetic rats on extracellular calcium availability for clonidine-induced contractile force generation. These data indicate that the increased noradrenaline-induced responsiveness of aortae from diabetic rats may be attributed to an increased alpha 2-adrenoreceptor component of the contractile response. It is also concluded that the alpha 2-adrenoreceptor mediated contraction is primarily dependent upon extracellular calcium. From this we suggest that the increased contractile responsiveness of aortae from diabetic rats to alpha 2-adrenoreceptor stimulating drugs may be related to an altered coupling mechanism between the alpha 2-adrenoreceptors and the calcium ion channels or due to a change in membrane permeability as a result of the disease process.

Mesenteric arteries from streptozotocin (STZ)-diabetic rats developed greater contractile force in response to norepinephrine and related alpha-agonists than arteries from age-matched controls. Subsequent experiments attempted to define the mechanisms underlying these findings. Transmural nerve stimulation of mesenteric arteries from both groups of animals revealed a similar optimal frequency and voltage of stimulation; however, arteries from STZ-diabetic rats developed greater contractile force than controls. Second, determination of selective alpha-adrenergic antagonist affinities (pA2 values) revealed qualitatively similar postjunctional alpha 1-adrenoceptors in both groups of arteries. Third, disruption of the endothelium did not abolish the enhanced responsiveness of arteries from STZ-diabetic rats. In contrast, the increased vascular responsiveness in STZ-diabetes was associated with a greater dependency on extracellular calcium, with no change in the response to alpha-agonist-induced release of calcium from cellular stores. Thus the enhanced responsiveness of mesenteric arteries from STZ-diabetic rats to alpha-adrenergic agonists cannot be attributed to neuronal deterioration, altered postjunctional alpha-adrenoceptor subtypes, endothelium degeneration, or enhanced release of intracellular calcium but is associated with a greater dependency on extracellular calcium.

Diabetes mellitus is known to produce alterations in vascular reactivity. In the present study we have examined the effects of short-term diabetes on histamine-induced relaxation of isolated mesenteric arteries, and the role of the endothelial cell layer in this response. Removal of the endothelium completely abolished the histamine relaxation effect in both diabetic and age-matched control rats. In contrast, vessels isolated from streptozotocin-diabetic rats were supersensitive to histamine, and this relaxation was mediated only through the H1-receptors. The present findings suggest that histamine-induced relaxation of rat mesenteric arteries is dependent upon endothelial cell processes which are enhanced in arteries from STZ-diabetic rats.

Right atria from rats rendered diabetic by injection of streptozocin (STZ-D) for 8-10 wk are supersensitive to the negative chronotropic effects of muscarinic agonists but have decreased levels of muscarinic receptors and acetylcholinesterase activity. Insulin treatment completely prevents the development of these changes. The proportion of atrial muscarinic receptors displaying high-affinity agonist binding is lower in STZ-D rats; however, the sensitivity of high-affinity agonist binding to regulation by a guanine nucleotide (5'-guanylylimidodiphosphate) is greater in atria from diabetic rats. Again, insulin treatment eliminates these differences. These findings indicate that alterations in atrial muscarinic systems in STZ-D rats are a consequence of the elaboration of the diabetic state and suggest that an alteration of functional muscarinic receptor-G protein coupling contributes to the altered physiological responsiveness of the heart in diabetes.

The mechanism responsible for increased norepinephrine-induced responsiveness of aortas isolated from streptozotocin-diabetic rats compared to age-matched control animals was investigated. Selective alpha-1 and alpha-2 adrenoceptor agonists and antagonists were used to determine the contribution of these receptor subtypes to the norepinephrine-induced contractile response. Findings from these experiments indicated an enhancement of alpha-2 adrenoceptor-mediated contraction in aortas from diabetic rats, whereas alpha-1-mediated responses were not altered. The contribution of extracellular calcium influx to the agonist-induced contractions was determined by using the calcium entry blocker nifedipine. Contractile responses to maximally effective concentrations of norepinephrine, phenylephrine and clonidine were separated into fast and slow components and the effect of increasing concentrations of nifedipine on the responses was determined. These experiments indicated that the fast component of contraction to alpha adrenoceptor agonists was insensitive to nifedipine treatment, whereas the slow component was inhibited effectively. The slow component of contraction in response to norepinephrine and clonidine in aortas from diabetic rats was increased significantly compared to control tissues. These results suggest that there is an increase in alpha-2 adrenoceptor activity in aortas from diabetic rats. Furthermore, the increased aortic contractile responses induced by norepinephrine and selective alpha-2 agonists are due probably to an increased influx of extracellular calcium through nifedipine-sensitive ion channels associated with activation of alpha-2 adrenoceptors.

The cardiac cholinergic system was studied in streptozotocin (STZ)-diabetic and age-matched control rats. STZ-diabetic rats (8-10 weeks) were supersensitive to the negative chronotropic effects of acetylcholine, carbamylcholine and bethanechol; inotropic responses to these muscarinic agonists were unaltered. This phenomenon was associated with a decrease in acetylcholinesterase activity but no change in the rate and extent of neuronal choline uptake. [3H]N-methylscopolamine bound to muscarinic receptors in atria from both groups of rats with the same high affinity. The density of [3H]N-methylscopolamine binding sites, however, was 34% lower in atria from STZ-diabetic rats. Agonist binding affinity was lower in diabetes; carbamylcholine had a lower affinity for both the high- and low-affinity receptors. These results indicate that cardiac cholinergic supersensitivity in right atria in diabetes occurs before the development of autonomic neuropathy insofar as neuronal [3H]choline uptake is unaltered at this stage of STZ diabetes. Changes in agonist binding conformation, without a concomitant change in antagonist binding affinity, suggest that supersensitivity of right atria to muscarinic agonist may be a consequence of altered coupling of muscarinic receptor to transduction mechanisms involved in chronotropism in diabetes.

The purpose of the study was to examine changes in the contractile responses to norepinephrine of aortas obtained from streptozotocin-induced diabetic rats and to determine the effects of changes in extracellular calcium (Ca++) concentrations on those contractile responses. Aortic rings from rats diabetic 14 to 20 days were supersensitive to norepinephrine in 0.20, 0.40 and 0.80 mM Ca++, but not in 2.50 mM Ca++. Aortas from rats diabetic 28 to 35 days showed a marked increase in contractile force to norepinephrine in 0.20, 0.40, 0.80 and 2.50 mM Ca++. The contractile force was significantly greater in the 28 to 35 day diabetic group as compared to the 14 to 20 day diabetic group. When the Ca++ concentration was changed from 2.50 mM to 0.20 and 0.40 mM, the aortas from the 28 to 35 days diabetic rats showed a significant decrease in developed tension. These data indicate that vascular reactivity is altered in experimental diabetes, and this alteration is related to the duration of the diabetic state and to the extracellular Ca++ concentration.