Increased expression of adenylyl cyclase 3 in pancreatic islets and central nervous system of diabetic Goto-Kakizaki rats

Adenylyl cyclase 3 (AC3) is expressed in pancreatic islets of the Goto-Kakizaki (GK) rat, a spontaneous animal model of type 2 diabetes (T2D), and also exerts genetic effects on the regulation of body weight in man. In addition to pancreatic islets, the central nervous system (CNS) plays an important role in the pathogenesis of T2D and obesity by regulating feeding behavior, body weight and glucose metabolism. In the present study, we have investigated AC3 expression in pancreatic islets, striatum and hypothalamus of GK rats to evaluate its role in the regulation of glucose homeostasis. GK and Wistar rats at the age of 2.5 mo were used. A group of GK rats were implanted with sustained insulin release chips for 15 d. Plasma glucose and serum insulin levels were measured. AC3 gene expression levels in pancreatic islets, striatum and hypothalamus were determined by using real-time RT-PCR. Results indicated that plasma glucose levels in Wistar rats were found to be similar to insulin-treated GK rats, and significantly lower compared with non-treated GK rats. AC3 expression levels in pancreatic islets, striatum and hypothalamus of GK rats were higher compared with Wistar rats, while the levels were intermediate in insulin-treated GK rats. The AC3 expression display patterns between pancreatic islets and striatum-hypothalamus were similar. The present study thus provides the first evidence that AC3 is overexpressed in the regions of striatum and hypothalamus of brain, and similarly in pancreatic islets of GK rats suggesting that AC3 plays a role in regulation of glucose homeostasis via CNS and insulin secretion.     

Modification of Clonidine-Induced Pressor Responses in Morphine-Dependent and Hypophysectomised Rats

Pressor responses to successive doubling doses of clonidine were examined in anaesthetised normotensive (NT) and spontaneously hypertensive rats (SHR). The slope of the dose-response curves was similar in both strains of rat, but the average response to the same concentrations of clonidine was less in the SHR. Naloxone (2 mg/kg i.v.) did not alter the dose-response curve in either strain. However, in NT and SHR made morphine-dependent (3 × 75 mg morphine pellets s.c.) and in hypophysectomised rats the increase in blood pressure was significantly reduced compared to the untreated or sham operated control groups, with a decrease in both the slope of the dose-response curve and a decrease in the average response. These results suggest that opioid peptide systems can modify vascular reactivity although not via circulating peptides. The specificity of the effects remains to be established     

Contribution of the region anterior and ventral to the third ventricle to opiate withdrawal excitation of oxytocin secretion.

Virgin female or lactating rats were given infusion into a lateral cerebral ventricle (i.c.v.) of either morphine to produce tolerance and dependence or vehicle from a subcutaneous osmotic minipump for 5 days, then they were anaesthetized with urethane. In virgins either an electrolytic or sham lesion of the region anterior and ventral to the third ventricle (AV3V region) was made. Initial blood plasma concentrations of oxytocin, measured by radioimmunoassay were similar in i.c.v. morphine- and i.c.v. vehicle-infused rats (20.6 +/- 2.7 and 19.0 +/- 4.3 pg/ml, respectively). Naloxone (5 mg/kg i.v.) significantly increased oxytocin secretion in all groups for at least 60 min; oxytocin concentration at 6 min after naloxone was in the order: sham-lesioned i.c.v. morphine group (mean 1,839 +/- 809 pg/ml, n = 6), greater than AV3V-lesioned i.c.v. morphine group (326 +/- 65 pg/ml, n = 6), = sham-lesioned i.c.v. vehicle group (251 +/- 66 pg/ml, n = 6), greater than AV3V-lesioned i.c.v. vehicle group (47.2 +/- 12.4 pg/ml, n = 6). Thus in both intact and lesioned rats naloxone increased oxytocin secretion much more in morphine-dependent rats than in the respective controls; in both morphine-naive and morphine-dependent rats, the AV3V lesion reduced the effect of naloxone with respect to plasma oxytocin concentration, but not with respect to the increase relative to the lower prenaloxone concentrations in the lesioned rats (prenaloxone values in the lesioned rats were: i.c.v. vehicle group, 15.8 +/- 6.8 pg/ml; i.c.v. morphine group, 24.3 +/- 7.8 pg/ml; and in the sham-lesioned rats: i.c.v. vehicle group, 67.3 +/- 31.2 pg/ml, i.c.v. morphine group, 65.5 +/- 15.0 pg/ml). Thus the AV3V region is not essential for withdrawal excitation of oxytocin secretion in morphine-dependent rats. In lactating morphine-dependent rats, i.c.v. infusion of the angiotension II antagonist saralasin (2.5 micrograms/min) decreased plasma oxytocin concentration after 10 min (5.7 +/- 1.1 pg/ml, n = 7 vs. 13.2 +/- 3.2 pg/ml, n = 8), but did not prevent naloxone-provoked excitation of oxytocin secretion, measured by radioimmunoassay (6 min after naloxone in the i.c.v. saralasin group 402.8 +/- 124.5 pg/ml, n = 7 vs, in controls, 1,009 +/- 382 pg/ml, n = 7) or assessed by continuous recording of intramammary pressure. These results indicate that centrally acting angiotensin II is not an important mediator of naloxone-induced oxytocin hypersecretion in morphine-dependent rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cyclic Nucleotide Metabolism in Pineal Homogenates

Adenylate cyclase (AC) in pineal particulate fractions from rabbit, rat, cow, and the vole Microtus montanus was stimulated by L-norepinephrine (NE) and L-isoproterenol (ISO). NE stimulation of rabbit and bovine pineal AC was biphasic, with a plateau between 0.01 microM and 1.0 microM and additional stimulation by NE above 1.0 microM. Stimulation by different ISO concentrations gave a typical hyperbolic curve, and optimal stimulation by ISO exceeded that by NE. Melatonin decreased ISO and NE stimulation of AC 10-20%. Although, alpha-adrenergic agonists increase beta-agonist-mediated adenosine-3',5'-cyclic monophosphate (cyclic AMP) accumulation in intact pinealocytes, similar amplification of AC stimulation was not seen with broken-cell preparations. Most (60-70%) pineal guanylate cyclase (GC) was recovered in supernatant fractions after centrifugation of homogenates at 110,000 x g; this soluble GC was unaffected by potential agonists. Low concentrations (0.01-1 nM) of NE, ISO, and phenylephrine (PE) stimulated GC in impure and purified membrane fractions, but each inhibited at concentrations above 10 microM. All concentrations of ISO and NE inhibited GC in the presence of the alpha-agonist PE. Melatonin alone did not affect particulate GC, but L-ISO stimulation was not seen in the presence of equivalent concentrations of melatonin. The in vitro data are consistent with both alpha- and beta-receptor regulation of cyclic nucleotide metabolism in pinealocytes. Endogenous NE may differentially regulate cyclic AMP and guanosine-3',5'-cyclic monophosphate (cyclic GMP) in pineal; low NE concentrations that stimulate GC have only a slight effect on AC, but higher NE concentrations that inhibit GC maximally stimulate AC. Particulate GC and AC also were resolved by equilibrium centrifugation, to give several discrete peaks of enzyme activity. The results support the existence of several forms of AC and GC, which have different responses to adrenergic agonists.     

Increased expression of adenylyl cyclase 3 in pancreatic islets and central nervous system of diabetic Goto-Kakizaki rats: A possible regulatory role in glucose homeostasis

Adenylyl cyclase 3 (AC3) is expressed in pancreatic islets of the Goto-Kakizaki (GK) rat, a spontaneous animal model of type 2 diabetes (T2D), and also exerts genetic effects on the regulation of body weight in man. In addition to pancreatic islets, the central nervous system (CNS) plays an important role in the pathogenesis of T2D and obesity by regulating feeding behavior, body weight and glucose metabolism. In the present study, we have investigated AC3 expression in pancreatic islets, striatum and hypothalamus of GK rats to evaluate its role in the regulation of glucose homeostasis. GK and Wistar rats at the age of 2.5 mo were used. A group of GK rats were implanted with sustained insulin release chips for 15 d. Plasma glucose and serum insulin levels were measured. AC3 gene expression levels in pancreatic islets, striatum and hypothalamus were determined by using real-time RT-PCR. Results indicated that plasma glucose levels in Wistar rats were found to be similar to insulin-treated GK rats, and significantly lower compared with non-treated GK rats. AC3 expression levels in pancreatic islets, striatum and hypothalamus of GK rats were higher compared with Wistar rats, while the levels were intermediate in insulin-treated GK rats. The AC3 expression display patterns between pancreatic islets and striatum-hypothalamus were similar. The present study thus provides the first evidence that AC3 is overexpressed in the regions of striatum and hypothalamus of brain, and similarly in pancreatic islets of GK rats suggesting that AC3 plays a role in regulation of glucose homeostasis via CNS and insulin secretion.     

Regional cerebral glucose utilization during morphine withdrawal in the rat.

Regional cerebral glucose utilization was studied by 2-deoxy[14C]glucose autoradiography in morphine-dependent rats and during naloxone-induced morphine withdrawal. In morphine-dependent rats, glucose utilization was increased compared with naive controls uniformly (23-54%) in hippocampus, dentate gyrus, and subiculum and reduced in frontal cortex, striatum, anterior ventral thalamus, and medial habenular nucleus. On precipitation of morphine withdrawal by subcutaneous administration of naloxone at 0.5 mg/kg to morphine-dependent rats, glucose utilization was increased in the central nucleus of amygdala (51%), lateral mammillary nucleus (40%), lateral habenular nucleus (39%), medial mammillary nucleus (35%), and medial septal nucleus (35%) (all, P less than 0.01). Significant increases also occurred in several other limbic structures including interpeduncular nucleus, anterior medial and ventral thalamic nuclei, and lateral septal nucleus. Knowledge of the functional cerebral anatomy of the morphine-withdrawal syndrome should facilitate studies directed toward understanding the molecular mechanisms of opiate withdrawal.     

Effects of adrenalectomy and thyroidectomy on postnatal rat lung development and cytoplasmic factors modulating adenylate cyclase activity

Effects of adrenalectomy (ADX) and thyroidectomy (THX) on postnatal rat lung growth and development were investigated in relation to the cytoplasmic factors modulating adenylate cyclase activity. On day 21 ADX and THX in rats reduced body and lung weights on days 31 and 43. However, lung weight/body weight was not affected by ADX or THX. Treatment of ADX and THX rats with dexamethasone and thyroxine (T₄), respectively, for 10 days neither restored body growth to the control level nor had any effect on lung growth. Neither protein, DNA, or protein/DNA showed any significant changes in lungs of ADX and THX rats. However, lung glycogen increased in ADX rats and decreased on treatment with dexamethasone. On the other hand, THX and treatment of THX rats with T₄ essentially had no effect on lung glycogen level. ADX resulted in lower adenylate cyclase activity and its stimulation by the cytoplasmic factors in ADX rat lungs. Dexamethasone treatment of ADX rats restored the basal adenylate cyclase activity to the control level, but potentiated the cytoplasmic activation of adenylate cyclase much more than the control lung supernatant. THX, on the other hand, increased the basal adenylase cyclase activity, but its stimulation by the lung supernatant was diminished. Treatment of THX rats with T₄ did not alter the basal adenylate cyclase activity compared with THX rats, but the cytoplasmic activation of adenylate cyclase was restored to the control level. Alterations in adenylate cyclase activation by the lung supernatant from ADX and THX rats did not appear to be due to changes in either the amount of enzyme in membranes or its sensitivity to the cytoplasmic factors. While ADX did not alter cyclic AMP-phosphodiesterase activity in rat lung, THX reduced this enzyme activity, which was restored to the control level on treatment of THX rats with T₄. These data suggest that hormones such as glucocorticoids and thyroxine may influence lung growth and development by inducing alterations in the cytoplasmic factors activating adenylate cyclase, cyclic AMP, and glycogen metabolism.     

Cardiac Adenylyl Cyclase and Phosphodiesterase Expression Profiles Vary by Age,Disease, and Chronic Phosphodiesterase Inhibitor Treatment

Background

Pediatric heart failure (HF) patients have a suboptimal response to traditional HF medications, although phosphodiesterase-3 inhibition (PDE3i) has been used with greater success than in the adult HF population. We hypothesized that molecular alterations specific to children with HF and HF etiology may affect response to treatment.

Noradrenergic and dopaminergic activity in the hypothalamic paraventricular nucleus after naloxone-induced morphine withdrawal

Previous research has shown an increase in hypothalamo-pituitary-adrenal axis activity following naloxone administration to morphine-dependent rats. In the present study, we investigated the adaptive changes in the noradrenaline (NA) and dopamine (DA) systems in the hypothalamic paraventricular nucleus (PVN) during morphine dependence and withdrawal. Additionally, we examined the possible change in 3',5'-cyclic adenosine monophosphate (cAMP) levels in that nucleus under the same conditions. Rats were made dependent on morphine by morphine or placebo (na?ve) pellet implantation for 7 days. On day 8, rat groups received an acute injection of saline or naloxone (1 mg/kg subcutaneously) and were decapitated 30 min later. NA and DA content as well as their metabolite production in the PVN were estimated by HPLC/ED. Both plasma corticosterone levels and cAMP concentration in the PVN were measured by RIA. Naloxone administration to morphine-dependent rats (withdrawal) induced a pronounced increase in the production of both the NA metabolite MHPG and the DA metabolite DOPAC and an enhanced NA and DA turnover. Furthermore, an increase in corticosterone secretion was observed in parallel to the changes in catecholamine turnover. However, no alterations in cAMP levels were seen during morphine withdrawal. These results raise the possibility that catecholaminergic afferents to the PVN could play a significant role in the alterations of PVN functions and consequently in the pituitary-adrenal response during morphine abstinence syndrome. These data provide further support for the idea of adaptive changes in catecholaminergic neurons projecting to the PVN during chronic morphine exposure.     

Ectopic expression of guanylyl cyclase C in gastric cancer as a potential biomarker and therapeutic target

OBJECTIVE: To investigate the expression of guanylyl cyclase C (GCC) in human gastric cancer (GC) tissues and assess the effect of GCC small interfering RNA (siRNA) on the proliferation and apoptosis of SGC‐7901. METHODS: The expression of GCC in 30 specimens and three human GC cell lines (SGC‐7901, AGS, NCI‐N87) were detected by RT‐PCR for messenger RNA (mRNA) by Western blot and immunofluorescence for proteins. Recombinant plasmids containing GCC siRNA and scrambled siRNA were constructed and transfected into SGC‐7901 cells, respectively. A cell counting kit‐8, flow cytometry (FCM) and terminal deoxynucleotidyl transferase (TDT)‐mediated dUTP‐biotin nick end‐labeling were used to evaluate cell viability, cell cycle distribution and apoptosis, followed by wound healing assay and cell adherent assay for cell motility and adherent, respectively. RESULTS: The expression of GCC was absent in paracancerous tissues, whereas the GCC mRNA and protein expressions were detected in 20/30 and 19/30 of GC specimens, respectively. Moreover, intestinal GC was statistically different from diffuse GC (P < 0.05). The proliferation of SGC‐7901 cells was markedly inhibited by GCC siRNA‐3 (P < 0.05) and cell morphological changes including volumetric reduction, karyopyknosis and karyorrhexis were observed. FCM showed that the cell count in the sub‐G0/G1 peak increased from 5.47% (48 h after transfection) to 5.63% (72 h after transfection). The wound healing assay and cell adherent assay revealed that GCC gene silencing decreased cell motility and adherent. CONCLUSION: The over‐expression of GCC has been detected in intestinal type GC. GCC siRNA can effectively inhibit the proliferation and invasion of SGC‐7901 cells and induce cell apoptosis. GCC might be a novel biomarker and therapeutic target for GC.     

The Cardiac β -Adrenoceptor-G-protein(s)-adenylyl Cyclase System in Monocrotaline-treated Rats

In rats, injection of the alkaloid monocrotaline (MCT) causes right ventricular hypertrophy and cardiac failure. In order to study whether, in MCT-treated rats, changes in the cardiac β -adrenoceptor-G-protein(s)-adenylyl cyclase system might be comparable to those found in human primary pulmonary hypertension, we assessed in right and left ventricles from MCT-treated rats the components of the β -adrenoceptor system: the receptor number and subtype distribution (by (-)-[¹²⁵I]iodocyanopindolol binding), the G-proteins (by quantitative Western blotting), and the activity of adenylyl cyclase. A single injection of 60 mg/kg i.p. MCT caused in rats right ventricular hypertrophy (RVH); part of the rats developed cardiac failure (RVF). In these rats the cardiac β -adrenoceptor-G-protein(s)-adenylyl cyclase system was markedly changed β -adrenoceptors were desensitized due to a decrease in receptor number, an uncoupling of the receptor from the G_s-adenylyl cyclase system, a decrease in G_sand a decrease in the activity of the catalytic unit of adenylyl cyclase. In general, these changes were more pronounced in right ventricles v left ventricles, and in rats with RVF v rats with RVH. On the other hand, cardiac muscarinic receptors and G_iappeared not to be altered. We conclude that in MCT-treated rats changes in the cardiac β -adrenoceptor-G-protein(s)-adenylyl cyclase system occur that resemble those observed in human primary pulmonary hypertension. Thus, MCT-treated rat appears to be a suitable animal model to study in more detail the pathophysiology of the development of right heart failure, and to identify new therapeutic possibilities.     

Mechanisms underlying the effects of estrogen on nocturnal melatonin synthesis in peripubertal female rats: relation to norepinephrine and adenylate cyclase

We previously reported that estrogen modulates the nocturnal synthesis of melatonin in the pineal gland of peripubertal female rats. These effects appeared to be mediated by the modulation of N-acetyltransferase (NAT) activity. The present study assessed the mechanism underlying the effects of estrogen deficiency and stimulation on pineal melatonin synthesis in peripubertal female rats. We measured the norepinephrine levels and adenylate cyclase activity in pineal gland homogenates obtained from 4-10-wk-of-age female Sprague Dawley rats at mid-dark during the daily light/dark cycle. The animals were ovariectomized and daily s.c. administration of estradiol benzoate (E2B, 1.0 microgram/d) was initiated at 4 wk of age. Pineal norepinephrine levels increased significantly from Week 3 to 4 (P < 0.0001), and remained unchanged thereafter. Neither ovariectomy nor E2B administration significantly affected norepinephrine levels. Adenylate cyclase activity in the pineal gland peaked at 4 wk in untreated (control) rats. Ovariectomy at Week 4 led to a significant increase in adenylate cyclase activity at Week 8. At Week 10, adenylate cyclase activity returned to control levels. S.c. injection of E2B suppressed the ovariectomy-induced increase in adenylate cyclase activity to the level seen in control rats. These changes in mid-dark adenylate cyclase activity resembled those previously observed with NAT activity. The results suggest that estrogen modulates adenylate cyclase activity in the pineal gland of peripubertal female rats. The inhibitory effect of estrogen on melatonin synthesis appeared to be mediated in part, by changes in the norepinephrine-induced stimulation of pineal adenylate cyclase activity.     

Desensitization of the adipocyte A(1) adenosine receptor during untreated experimental diabetes mellitus

We examined the changes that occur in the adenosine receptor system during diabetes mellitus. Experimental diabetes mellitus was induced in male Lewis rats with streptozocin (65 mg/kg), and A₁ adenosine receptor binding was characterized with [¹²⁵I]N ⁶-2-(4-aminophenyl) ethyladenosine. In adipocytes, high-affinity A₁ adenosine receptor binding decreased from 1466±228 of protein to 312±123 fmol/mg of protein (p<0.01) following 14 d of untreated diabetes mellitus. Neither the dissociation constant (K _d=1.3±0.2 nM) nor the basal level of adenylate cyclase activity (2.8±1.1 pmol cAMP/mg of protein/min) was altered by diabetes mellitus. The dose-response curve for the inhibition of adenylate cyclase byN ⁶-R-phenylisopropyladenosine (R-PIA), however, did show a rightward shift, indicating that diabetic adipocyte membranes were less sensitive to the effects of adenosine than nondiabetic adipocyte membranes. In contrast, the A₁ adenosine receptor-binding characteristics and adenylate cyclase dose-response curve for cerebral cortical tissue were unchanged by diabetes. These findings suggest that diabetes has tissue-specific effects on the A₁ adenosine receptor system. Furthermore, the decreased sensitivity to adenosine potentially worsens the hyperlipidemia associated with diabetes mellitus. Such alterations in the adenosine receptor system may play a previously undescribed role in the pathophysiology of diabetes mellitus and may help explain why some organs are severely affected by diabetes, but others are relatively spared. Understanding these alterations in adenosine receptor function may lead tonovel therapies of this common metabolic disease.     

Release of oxytocin but not corticotrophin-releasing factor-41 into rat hypophysial portal vessel blood can be made opiate dependent

The effects of morphine dependence and abrupt opiate withdrawal on the release of oxytocin and corticotrophin-releasing factor-41 (CRF-41) into hypophysial portal vessel blood in rats anaesthetized with urethane were investigated. Adult female Sprague-Dawley rats were made dependent upon morphine by intracerebroventricular infusion of morphine for 5 days; abrupt opiate withdrawal was induced by injection of the opiate antagonist naloxone. The basal concentrations of oxytocin in portal or peripheral plasma from morphine-dependent rats did not differ significantly from those in control, vehicle-infused rats. In rats in which the pituitary gland was not removed after stalk section, the i.v. injection of naloxone hydrochloride (5 mg/kg) resulted in a large and sustained increase in the concentration of oxytocin in both portal and peripheral plasma in control and morphine-dependent rats. The i.v. injection of naloxone resulted in a threefold increase in the secretion of oxytocin into portal blood in acutely hypophysectomized rats infused with morphine, but did not alter oxytocin secretion in vehicle-infused hypophysectomized rats. The concentration of oxytocin in peripheral plasma in both vehicle- and morphine-infused hypophysectomized rats was at the limit of detection of the assay and was unchanged by the administration of naloxone. There were no significant differences in the secretion of CRF-41 into portal blood in vehicle- or morphine-infused hypophysectomized rats either before or after the administration of naloxone. These data show that, as for oxytocin release from the neurohypophysis into the systemic circulation, the mechanisms which regulate oxytocin release into the portal vessel blood can also be made morphine dependent. The lack of effect of morphine or naloxone on the release of CRF-41 or other stress neuro-hormones suggests that the effect of opiate dependence and withdrawal is selective for oxytocin and is not simply a non-specific response to 'stress'.     

Type VIII adenylyl cyclase in rat beta cells: coincidence signal detector/generator for glucose and GLP-1

Aims/hypothesis The secretory function of pancreatic beta cells is synergistically stimulated by two signalling pathways which mediate the effects of nutrients and hormones such as glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic peptide (GIP) or glucagon. These hormones are known to activate adenylyl cyclase in beta cells. We examined the type of adenylyl cyclase that is associated with this synergistic interaction.Methods Insulin release, cAMP production, adenylyl cyclase activity, mRNA and protein expression were measured in fluorescence-activated cell sorter-purified rat beta cells and in the rat beta-cell lines RINm5F, INS-1 832/13 and INS-1 832/2.Results In primary beta cells, glucagon and GLP-1 synergistically potentiate the stimulatory effect of 20 mmol/l glucose on insulin release and cAMP production. Both effects are abrogated in the presence of the L-type Ca²⁺-channel blocker verapamil. The cAMP-producing activity of adenylyl cyclase in membranes from RINm5F cells is synergistically increased by Ca²⁺-calmodulin and recombinant GTPS-activated G_s-protein subunits. This type of regulation is characteristic for type I and type VIII AC isoforms. Consistent with this functional data, AC mRNA analysis shows abundant expression of type VI AC, four splice variants of type VIII AC and low expression level of type I AC in beta cells. Type VIII AC expression at the protein level was observed using immunoblots of RINm5F cell extracts.Conclusion/interpretation This study identifies type VIII AC in insulin-secreting cells as one of the potential molecular targets for synergism between GLP-1 receptor mediated and glucose-mediated signalling.Abbreviations AC Adenylyl cyclase - G_s alpha subunit of G-protein stimulatory to adenylyl cyclase - AKAP A-kinase anchoring protein - GLP-1 glucagon-like peptide 1 - GIP glucose-dependent insulinotropic peptide - IBMX 3-isobutyl-1-methylxanthine - PDE phosphodiesterase - PKA protein kinase A - PP2B protein phosphatase 2B     

Free radicals generated by xanthine oxidase-hypoxanthine damage adenylate cyclase and ATPase in gerbil cerebral cortex

The generation of superoxide radicals from xanthine oxidase-hypoxanthine in a particulate fraction of gerbil cerebral cortex influenced the activity of the synaptic enzyme adenylate cyclase, as well as Mn²⁺- and Na⁺,K⁺-sensitive forms of ATPase. Low concentrations of xanthine oxidase actually elevated the sensitivity of adenylate cyclase to GTP, GTP + norepinephrine (NE), and forskolin but not significantly to Mn²⁺. Higher levels of xanthine oxidase elicited a marked inhibition of these responses. The stimulation of adenylate cyclase mechanisms requiring GTP (GTP, forskolin, and NE) was more susceptible than was Mn²⁺, suggesting that the guanine nucleotide stimulatory protein was more vulnerable to free radical attack than the catalytic site of adenylate cyclase. superoxide dismutase (SOD), but not catalase, partially protected the forskolin-sensitive enzyme from the action of xanthine oxidase-hypoxanthine. A combination of SOD plus catalase preserved enzyme responses to forskolin. In comparison, additions of SOD plus mannitol or catalase plus flunarizine were less effective. The sensitivity of the particulate ATPase to Mn²⁺ was more labile to the consequence of superoxide formation than Na⁺, K⁺-ATPase. In this regard the Ca²⁺, Mg²⁺ sensitivity of the enzyme was reduced only to a marginal extent. The findings might be analogous toin vivo data in which cerebral adenylate cyclase and Na⁺, K⁺-ATPase are damaged following postischemic reperfusion in gerbils, a process thought to be mediated by free radicals.     

Transgenerational modification of hippocampus TNF-α and S100B levels in the offspring of rats chronically exposed to morphine during adolescence

Background: TNF-α and S100B are important signaling factors that are involved in many aberrant conditions of the brain. Chronic morphine exposure causes aberrant modifications in the brain. Objectives: We examined the consequences of chronic morphine consumption by parents before mating on hippocampus TNF-α and S100B levels in the parents and their offspring. Methods: A total of 12 adult female and 12 adult male Wistar rats were used as parents. Each gender was divided randomly into two groups: control and morphine consumer. Morphine consumer groups received morphine sulfate dissolved in drinking water (0.4 mg/ml) for 60 days. Control groups received water. Thirty days before mating, morphine was replaced with water. All offspring also received water. The hippocampus of both parental and offspring groups was extracted to measure TNF-α and S100B levels using an ELISA. Results: Hippocampus TNF-α levels were significantly increased due to chronic morphine use in both male and female parents compared to those of control parents (P < 0.01). Moreover, both male and female offspring of morphine-exposed parents showed a significant increase in hippocampus TNF-α levels compared to those of control offspring (P < 0.01). Hippocampus levels of S100B were significantly decreased in male (P < 0.05) but not female morphine consumer parents relative to control parents. Both male and female offspring of morphine-exposed parents showed significant decreases in hippocampus S100B levels (P < 0.05) compared to those of control offspring. Conclusions: The consequences of chronic morphine use by parents, even when it is stopped long before mating and pregnancy, could induce modifications in the hippocampus of the next generation.     

Influence of glucocorticoid on bone in 3-, 6-, and 12-month-old rats as determined by bone mass and histomorphometry

Abstract

The influence of glucocorticoid (GC) on bone in rats at different ages was investigated in order to provide insight into human glucocorticoid induced osteoporosis (GCOP). Three-, 6-, and 12-month-old female Wistar rats were divided into four groups: Zero-time control (ZT), vehicle (Cont), prednisolone (PSL) 2 mg/kg (P–L), PSL 20 mg/kg (P–H). PSL was subcutaneously administered every day for 4 weeks. Bone mineral density (BMD) at the proximal metaphysis and diaphysis of the tibia was measured by peripheral quantitative computed tomography. Histomorphometry of the tibia was performed for 3- and 6-month-old rats. GC increased trabecular and cortical BMD at the metaphysis in all 3-month-old rats with time. Trabecular BMD at the metaphysis in the P–L and P–H groups was significantly higher than in the control group. Histomorphometric parameters for both bone formation and resorption were also increased by GC treatment. In the 6-month-old rats, the metaphyseal trabecular BMD did not significantly change in any group, but the diaphyseal trabecular BMD significantly increased in the control group with time. The trabecular BMD of the metaphysis and diaphysis was significantly lower in the P–L and P–H groups than in the control group at week 4. Histomorphometric parameters for bone formation and resorption were both reduced by GC treatment. The BMD remained unchanged in all 12-month-old rats. Six-month-old rats treated with 20 mg/kg GC are suitable models for GC-induced osteoporosis with dominant cancellous bone decrease and reduced bone turnover. The pathology induced by 20 mg/kg prednisolone in 6-month-old female rats seems to be most similar to glucocorticoid-induced osteoporosis in humans.     

Altered action of glucagon on human liver in Type 2 (non-insulin-dependent) diabetes mellitus

Summary Glucagon may play a role in the metabolic derangements of overt Type 2 (non-insulin-dependent) diabetes mellitus. We therefore have evaluated the early steps in glucagon action by investigating the hormone-sensitive adenylyl cyclase system in liver membranes from seven Type 2 diabetic patients with fasting hyperglycaemia and two-fold elevations in plasma glucagon. The comparison was made with seven control subjects matched for age, sex and body weight. Glucagon receptor binding was almost identical in the two groups. There were, however, marked alterations in the adenylyl cyclase activity in membranes from the diabetic patients. This activity was reduced by 35–50% when compared to control activity. Basal cyclase activity, as well as the activity after stimulation with glucagon or with agents (i. e., sodium fluoride and forskolin) that act beyond the glucagon receptor, was significantly decreased (p<0.05, p<0.001 respectively). In conclusion, uncontrolled Type 2 diabetes in associated with an over-all loss of responsiveness of the hormone-sensitive adenylyl cyclase in human liver, which apparently results from post-receptor alterations. This change may provide a mechanism for reducing the effect of hyperglycagonaemia in Type 2 diabetes mellitus.