Decreased dynorphin levels and increased kappa opioid receptor binding in male rats with isolation-induced hypertension.

The role of hippocampal dynorphin A (1-8) (Dyn A (1-8)) and kappa opioid receptors was investigated in the isolation-induced hypertensive rat (IHR). Male Sprague Dawley rats were either isolated (1 per cage) or grouped (3 per cage) for 7 days. Isolated rats exhibited increased blood pressure (systolic blood pressure 159.7 +/- 6.6 mmHg) whereas the grouped rats remained normotensive (systolic blood pressure 137 +/- 6.3 mmHg). Using radioligand binding techniques we observed a significant increase in kappa opioid receptor binding in the hippocampus of isolated rats (56% increase) and this further increased when the length of isolation was increased to 2 weeks (72% increase). Radioimmunoassay showed that isolation decreased the hippocampal content of Dyn A (1-8) from 12.7 +/- 0.4 to 11.6 +/- 0.2 pg Dyn A (1-8) per 10 mg tissue (rats weighing approximately 100 g) and from 13.3 +/- 0.8 to 9.7 +/- 1 pg Dyn A (1-8) per 10 mg tissue (approximately 200 g rats). These data suggest that functional alterations in the hippocampal dynorphin system may be involved in the maintenance of isolation induced hypertension.

Kappa-opioid receptor antisense oligonucleotide injected into rat hippocampus causes hypertension.

Bi-hippocampal microinjection treatment (1 microg per side, twice a day for 5 days) with an antisense phosphorothioate oligodeoxynucleotide antisense oligodeoxynucleotide to the rat kappa-opioid receptor, caused hypertension in normotensive Wistar Kyoto (WKY) rats and increased the blood pressure of spontaneously hypertensive rats (SHR). Systolic blood pressure in WKY rats increased from 121+/-4 to 153+/-6 mm Hg, and in SHR systolic blood pressure increased from 153+/-4 to 183+/-5 mm Hg. Similar results were observed with mean blood pressure, however, there were no changes in heart rate. No significant responses were seen with either vehicle or missense injections. Radioligand binding studies indicated that there was a significant decrease in apparent kappa-opioid receptor density due to antisense oligodeoxynucleotide treatment. The results are in accord with our earlier suggestions that the kappa-opioid system in the hippocampus may have a role in the neural control of blood pressure.

Practical considerations of the pharmacology of angiotensin receptor blockers.

A review of the drug class of angiotensin receptor blockers (ARBs) as well as the ARBs currently available by prescription in the United States is presented. The importance of angiotensin II production by non-angiotensin-converting enzyme (non-ACE) pathways, particularly human chymase, is discussed. Emphasis is placed on the mechanism of action of ARBs and the different binding kinetics of these agents. Although all ARBs, as a group, block the AT1 receptor, they may differ in the pharmacological characteristics of their binding and be classified as either surmountable or insurmountable antagonists. Mechanisms of surmountable and insurmountable antagonism as well as possible benefits of these blocking characteristics are discussed in relation to the various ARBs. The cardiovascular effects of activation of the two main subtypes of angiotensin receptors (AT1 and AT2) are presented. In addition to their treatment of hypertension, ACE inhibitors are recognized as being effective in the management of heart failure, left ventricular hypertrophy, recurrent myocardial infarctions, and renal disease. ARBs are currently indicated only for the treatment of hypertension; however, in vitro and in vivo pharmacological studies as well as preliminary clinical data suggest that ARBs, like ACE inhibitors, may also provide effective protection against end-organ damage in these conditions.

Effects of rat brain kappa 1- and kappa 2-opioid receptors after chronic treatment with non-peptide kappa-agonists.

Injection of kappa-agonist dynorphins and non-peptide kappa-agonists into the hippocampus induces a reduction in blood pressure. It has been postulated that kappa-opioid agonists and kappa-receptors are important in one mechanism of antihypertension and might have clinical potential for the treatment of hypertension. We have investigated whether chronic treatment with U-50488H and U-62066E, two non-peptide kappa-agonists, effects brain kappa 1- or kappa 2-receptor numbers or affinities in areas that might correlate with changes in blood pressure. kappa 1- and kappa 2-Opioid receptor affinities and densities were determined in cortex, hippocampus, hypothalamus, midbrain and pons after 14 days subcutaneous infusion of two non-peptide kappa-agonists, U-50488H and U-62066E, 9.6 mg kg day-1, by means of osmotic minipumps, to spontaneously hypertensive rats (SHR) and to Wistar-Kyoto (WKY) rats. This infusion significantly reduced blood pressure. Brains were removed within 48 h of the end of drug infusion and kappa-receptor binding studies were performed on homogenates from each brain area using [3H]U-69593 to assay kappa 1-receptors and [3H]bremazocine to assay kappa 2-receptors. U-62066E treatment seemed to cause a slight decrease in the number of [3H]bremazocine binding sites (kappa 2-receptors) from 98.2 +/- 9 to 74.9 +/- 8 fmol (mg protein)-1 in the hippocampus when compared with SHR controls. A small decrease in kappa 2-receptor density in the pons of WKY rats was also observed after U-50488H treatment (control, 51.2 +/- 5; U-50488H-treated, 24.3 +/- 9 fmol (mg protein)-1. Although SHR blood pressure values were consistently reduced by treatment with kappa-agonists, there was little if any significant change in apparent numbers of kappa 1- or kappa 2-receptors or their affinities in any of the brain regions examined. These data indicate that although chronic treatment with kappa-agonists reduces blood pressure in SHR, the treatment does not elicit major changes in brain kappa-receptors either in SHR or in WKY rats. The potential use of kappa-agonists for treating hypertension might not cause receptor changes in the brain and might, therefore, result in fewer side effects or negligible rebound hypertension.

Sex hormones change adrenoceptors in blood vessels of the spontaneously hypertensive rat.

In this study we investigated how male sex hormones, which increase blood pressure in the spontaneously hypertensive rat (SHR), affect adrenergic receptors in the cardiovascular system. Testosterone treatment significantly increased blood pressure in male SHRs (P < 0.05). Testosterone treated male SHRs also showed a significant increase (P < 0.05) in total apparent numbers of alpha 1 adrenoceptors in tail artery preparations as compared to controls. Gonadectomy attenuated blood pressure and caused a decrease in the total apparent number of alpha 1 adrenoceptors in tail artery preparations (P < 0.05). Testosterone replacement therapy in these gonadectomized rats reversed this decrease in apparent number of alpha 1 adrenoceptors to control values. KD values for dihydroalprenolol, isoproterenol, prazosin and norepinephrine were not significantly different between treatment groups. These results indicate that sex hormones (androgens) modulate numbers of alpha 1 adrenoceptors in the cardiovascular system of the male SHR.

Dynorphin receptor changes in hippocampus of the spontaneously hypertensive rat.

Dynorphin receptor binding sites in hippocampal membrane preparations were assessed in spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats at 4, 8, 12 and 16 weeks of age. At 4 weeks of age, before hypertension is manifested, SHRs had significantly more hippocampal dynorphin receptor binding sites than WKY controls. At 8, 12 and 16 weeks of age, however, when hypertension is seen, SHRs showed significantly fewer hippocampal binding sites than WKY rats. No receptor affinity changes for dynorphin were seen between the two strains of rats at any age. These results suggest that hippocampal receptor changes involving the opioid system may play a role in the central component of blood pressure control.

Long-term consequences of prenatal exposure to cocaine or related drugs: effects on rat brain monoaminergic receptors.

Reports from both this laboratory and others indicate that prenatal exposure of rats to cocaine can produce alterations in development, activity and responses to environmental stimuli. In order to determine a biochemical basis for these effects, radioligand receptor-binding assays for different monoaminergic receptors were performed on rat brain tissues obtained from offspring of dams treated SC with saline, cocaine (15 mg/kg b.i.d.), amitriptyline (10 mg/kg) or amfonelic acid (AFA, 1.5 mg/kg). Male rat pups were fostered by surrogate dams and one rat per litter taken at 30, 60 or 180 days postnatal for determination of striatal and prefrontal cortical D2 receptors, prefrontal cortical 5HT2 receptors, cortical alpha 1-, alpha 2-, beta 1- and beta 2-adrenoceptors. Across all drug treatments and times, the only significant change was at 30 days of age when beta 1-adrenoceptors were increased 68% in the cocaine exposed pups--a time when these rats show hyperactivity--and at 180 days postnatal when a 20% decrease in DA2 receptor Bmax was observed. Also, cortical membrane Mg(2+)-dependent Na+, K(+)-ATPase activities and basal ATPase activities were unaltered by any of the treatments at any of the times. These results suggest that few changes have occurred in monoaminergic receptor sensitivity as a result of the exposure to these drugs during gestation. The behavioral changes that are known to occur following prenatal exposure to cocaine may be due to presynaptic alterations in neurotransmitter function rather than changes in postsynaptic receptors.

Effects of microgravity on brain neurotransmitter receptors.

Neurotransmitter receptor binding and Na+, K+-ATPase activity were examined in the brains of six rats exposed to 7 days of microgravity during the flight of Spacelab 3. The same variables were examined in a group of six ground control rats. 5-HT1 receptor number in the hippocampus was significantly elevated by exposure to the microgravity environment, and cortical sodium-potassium pump activity was significantly depressed. A marginal depression in dopamine D-2 binding in the striatum was noted. Dopamine and 5-HT binding in a wide variety of other central regions, in addition to GABAA, muscarinic acetylcholine, adenosine A1, and opiate receptor binding, and adrenoceptor binding, was unaffected by microgravity exposure.

Characterization of adenosine binding sites in bovine testicular tissue using 8-cyclopentyl-1,3-[3H]dipropylxanthine.

The existence of specific adenosine binding sites in bovine testicular tissue was evaluated using the novel antagonist radioligand 8-cyclopentyl-1,3-[3H]dipropylxanthine ([3H]DPCPX). Saturation analysis revealed specific binding that was saturable at approximately 1 nM. Scatchard analysis indicated a single class of binding sites with a KD = 0.26 nM and a Bmax = 0.37 pmol/mg protein. Affinity profiles suggest an A1 subtype recognition site that is different from the classical A1 adenosine receptor. The results presented should prove useful in subsequent studies concerning heterogeneity among adenosine receptors and also aid in discerning the role of adenosine in reproduction.

The effects of estradiol and mestranol on alpha-adrenoceptors in select regions of the rat brain.

The role of estrogens in modulating the concentration of CNS alpha-adrenoceptors has not been elucidated nor has it been determined how different estrogenic compounds affect these receptors. In this study brain alpha-1 and alpha-2-adrenoceptor binding was measured in female rats treated with estradiol and/or the synthetic estrogen mestranol. Rats treated biweekly for 12 weeks with mestranol (50 micrograms/100 g b.wt.) had a significant reduction in the apparent number of alpha-2-adrenoceptors in the frontal cortex and nucleus tractus solitarius (NTS), while apparent numbers of both alpha-1 and alpha-2-adrenoceptors were depressed in the locus coeruleus. Estradiol treatment (50 micrograms/100 g b.wt.) caused a significant elevation in apparent alpha-1-adrenoceptor numbers in the NTS relative to control. Alpha-adrenoceptor numbers in the rostral and caudal hypothalamus were not affected by either steroid treatment. These results suggest that regulation of apparent numbers of alpha-1 and alpha-2-adrenoceptors in the CNS depends on the type of estrogen used for treatment.

Estrogen modulation of uterine adrenergic-cholinergic interaction: effects on vasoactivity and adrenergic receptors in the guinea pig.

The regulation of uterine blood flow (UBF) in the guinea pig was investigated by determining the effects of steroid-catecholamine interaction on guinea pig UBF in cyclic (Day 0 = estrus) and ovariectomized (OVX)-steroid treated females. In cyclic guinea pigs, parallel elevations in uterine weight, UBF, beta and alpha receptor levels were observed during the estrus period, whereas uterine norepinephrine (NE) levels were low. In contrast, all parameters remained at low levels except NE levels during the luteal phase of the cycle which remained elevated in both normal and OVX-oil treated animals. Estrogen (E1)-treated females showed elevated uterine weights, UBF, beta, and alpha receptor levels but low uterine NE concentrations. Combined progesterone-estrogen (P2E1) treatment caused similar changes but maintained tissue NE at control levels. In the exposed uterine artery preparation, the sequential administration of acetylcholine followed by NE application induced a marked elevation in UBF in OVX, E-1-treated animals which was blocked by phentolamine (10(-6) M). This phenomena could not be demonstrated in either oil- or progesterone-treated OVX guinea pigs. In uterine membrane preparations from ovariectomized guinea pigs pretreated with either oil or progesterone, methacholine (cholinergic agonist; 10(-6) M) failed to alter the affinity of the alpha receptor for NE. However, in uterine preparations from OVX, estradiol-treated guinea pigs, methacholine significantly (P less than .05) increased the affinity of the alpha receptor for NE.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of environment on GABA receptors in muricidal rats.

The influence of environment (isolation) on GABA receptor numbers ( [3H]-muscimol binding sites) and affinities was investigated in specific limbic areas known to be involved with the development of muricide. Olfactory bulbs of isolated rats were found to have identical numbers of [3H]-muscimol binding sites whether or not they were muricidal. However, in the olfactory bulbs of the aggregated animals a greater than two-fold increase was found in numbers of [3H]-muscimol binding sites in those rats that were muricidal. In the amygdala muricidal rats had a 32-34% increase in [3H]-muscimol binding sites over non-muricidal rats regardless of environment. In the septum non-muricidal rats had fewer [3H]-muscimol binding sites than muricidal rats and although the trend was evident, statistical vigor was seen only in the aggregated animals. Neither muricide nor isolation significantly influenced [3H]-muscimol binding numbers in the hypothalamus. GABA Ki values were examined in all brain regions and found to be the same in the isolated and aggregated animals whether or not they were muricidal. We concluded that environment appears to influence apparent GABA receptor numbers in the olfactory bulbs and septum whereas muricidal behavior correlates well with an increase in apparent number of GABA receptors in the amygdala. GABA receptors in the hypothalamus were not influenced by either environment or aggression.

Regulation of estrogen-induced uterine hyperemia and contractility in the guinea pig: cholinergic modulation of an alpha-adrenergic response.

The effects of estradiol (1 microgram: E-1) treatment on uterine hyperemia and uterine sensitivity to various biogenic compounds were evaluated in ovariectomized (OVX) animals treated with either sesame oil or E-1 for 3 days. The E-1 treatments induced significant elevations in uterine weight, blood flow, and alpha- and beta-receptor numbers as compared with oil-treated controls. In contrast, uterine norepinephrine (NE) levels were reduced in E-1-treated, OVX guinea pigs as compared with oil-treated controls. Uterine sensitivity and responsivity to NE (10(-6) M) and acetylcholine (ACH: 10(-8) M) were either comparable to, or enhanced, in E-1-treated animals as compared with controls. In particular, combined ACH-NE treatment induced a dramatic increase in contraction force in E-1-treated uteri as compared with uteri from oil-treated animals. The use of specific adrenergic alpha- (phentolamine: 10(-6) M) or beta- (propranolol: 10(-6) M) receptor blocking agents indicated that the estrogenic response was mediated via the alpha-adrenergic receptor complex. Since atropine (10(-8) M) effectively blocked the cholinergic accentuation of this uterine response, it is suggested that a cholinergic priming, or beta-receptor block, is necessary for the full expression of the alpha-adrenergic-mediated, estrogenic response in the guinea pig. The estrogen-associated increase in available alpha- and beta-receptors and depressed tissue NE levels probably account for both the hyperemic response and enhanced tissue sensitivity to biogenic compounds in the guinea pig.

Clonidine and glucose intolerance.

Changes in carbohydrate metabolism were observed in a diabetic, hypertensive patient managed with clonidine. After a slight increase in the clonidine dose, his blood sugar control deteriorated. However, when the clonidine was withdrawn, the glucose intolerance subsided. Because clonidine preferentially binds alpha 2-subtype receptors, we investigated animal pancreatic tissue by radioligand binding technique and found it to contain alpha-adrenergic receptors predominantly of the alpha 2-subtype. In consideration of the response from withdrawal of clonidine and the results of our radioligand studies, wer concluded the glucose intolerance seen in this patient was most likely due to the specific action of clonidine on alpha 2-pancreatic receptors.

Myocardial adrenergic receptors and adenylate cyclase in the developing spontaneously hypertensive rat.

Cardiac membrane preparations from developing spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats (0 to 125 days of age) were analyzed for the apparent numbers of alpha- and beta-adrenergic receptors and adenylate cyclase activities in an attempt to correlate biochemical changes with the reported functional changes occurring with the development of hypertension in the SHR. Although the apparent number of alpha- and beta-adrenergic receptors were similar in both strains of rats, isoproterenol-stimulated adenylate cyclase activities were significantly higher (P less than 0.05) in the prehypertensive SHRs when compared to WKY rats and declined to lower values as hypertension appeared. The percent stimulation produced by isoproterenol remained similar in cardiac membranes from normotensive WKY rats at all ages of development whereas this percent stimulation was 40% higher at birth in the SHRs and declined to approximately one half the original value by 100 days of age (P less than 0.05). The elevated adenylate cyclase activity observed during the prehypertensive state may contribute to the genesis of hypertension.

Separation of vesicles of cardiac sarcolemma from vesicles of cardiac sarcoplasmic reticulum. Comparative biochemical analysis of component activities.

Sarcolemmal and sarcoplasmic reticulum membrane vesicle fractions were isolated from cardiac microsomes. Separation of sarcolemmal and sarcoplasmic reticulum membrane markers was documented by a combination of correlative assay and centrifugation techniques. To facilitate the separation, the crude microsomes were incubated in the presence of ATP, Ca2+, and oxalate to increase the density of the sarcoplasmic reticulum vesicles. After sucrose gradient centrifugation, the densest subfraction (sarcoplasmic reticulum) contained the highest (K+,Ca2+)-ATPase activity and virtually no (Na2+,K+)-ATPase activity, even when latent (Na+,K+)-ATPase activity was unmasked. In addition, the sarcoplasmic reticulum fraction contained no significant sialic acid, beta receptor binding activity, or adenylate cyclase activity. Sarcolemmal membrane fractions were of low buoyant density. Preparations most enriched in sarcolemmal vesicles contained the highest level of all the other parameters and only about 10% of the (K+,Ca2+)-ATPase activity of the sarcoplasmic reticulum fraction. The results suggest that (Na+,K+)-ATPase, sialic acid, beta-adrenergic receptors, and adenylate cyclase can be entirely accounted for by the sarcolemmal content of cardiac microsomes. Gel electrophoresis of the sarcolemmal and sarcoplasmic reticulum membrane fractions showed distinct bands. Membrane proteins exclusive to each of the fractions were also demonstrated by phosphorylation. Cyclic AMP stimulated phosphorylation by [gamma-32P]ATP of two proteins of apparent Mr = 20,000 and 7,000 that were concentrated in sarcoplasmic reticulum, but the stimulation was markedly dependent on the presence of added soluble cyclic AMP-dependent protein kinase. Cyclic AMP also stimulated phosphorylation of membrane proteins in sarcolemma, but this phosphorylation was mediated by an endogenous protein kinase activity. The apparent molecular weights of these phosphorylated proteins were 165,000, 90,000, 56,000, 24,000, and 11,000. The results suggest that sarcolemma may contain an integral enzyme complex, not present in sarcoplasmic reticulum, that contains beta-adrenergic receptors, adenylate cyclase, cyclic AMP-dependent protein kinase, and several substrates of the protein kinase.