Effects of somatostatin on dopamine sensitive adenylate cyclase activity in the caudate-putamen of the rat

Summary The effect of somatostatin-14 (SRIF) on dopamine-sensitive adenylate cyclase in caudateputamen pellets was studied in naive female rats, and in rats with chemical lesions of the nigrostriatal dopaminergic tract produced by injecion of 6-hydroxydopamine, or of the caudate-putamen itself produced by injection of kainic acid 3 week earlier. In unlesioned rats somatostatin at a concentration of 10^–7 moles/1 inhibited adenylate cyclase activation by submaximal concentrations of dopamine, increasing the apparent K_m but not altering E_max. In 6-hydroxydopamine lesioned rats somatostatin no longer influenced adenylate cyclase activity, whereas in kainic acid lesioned rats somatostatin still increased the apparent K_m for dopamine activation. The effect of somatostatin in untreated and lesioned rats is compatible with a partial competitive antagonism to dopamine. Although the data from the lesioned rats present preliminary results, the dose response characteristics and the effects in lesioned animals suggest a more complex interaction, possibly by binding of somatostatin to an inhibitory subunit of regulatory adenylate cyclase components.     

The effect of vasoactive intestinal polypeptide (VIP) on forskolin stimulated adenylate cyclase in the caudate-putamen of the rat

Summary Vasoactive intestinal polypeptide (VIP) was incubated in an adenylate cyclase assay with a particulate fraction of caudate-putamen (CP) tissue of the rat in order to examine the effect of the peptide on forskolin-activated adenylate cyclase in vitro. Forskolin induced an enhancement of cyclic AMP formation that was mediated by an effect on catalytic subunit and stimulatory guanine nucleotide regulatory protein (N_s). In our preparation, VIP did not influence basal adenylate cyclase activity or the stimulation by dopamine and sodium fluoride but, in the absence of guanylylimidodiphosphate (guanosine 5-(,y-imido)-triphosphate) VIP inhibited the forskolin-stimulation of the enzyme in a noncompetitive manner. Met-encephalin, acting on a D-2 receptor-coupled putative inhibitory guanine nucleotide regulatory protein (N_i), inhibited the adenylate cyclase activity stimulated by forskolin to a slightly greater extent than VIP. When assayed together, these inhibition effects were additive, implying that the peptide receptors are not identical. The N_i — antagonist, MnCl₂ completely blocked the inhibition of met-encephalin but had no significant effect on VIP-induced inhibition. In addition, pertussis toxin did not influence the effect of VIP on forskolin-stimulation in contrast to cholera toxin which did antagonize the VIP effect via the stimulatory guanine nucleotide regulatory protein (N_s). Furthermore, specific D-1 and D-2 dopaminergic receptor antagonists (+)-flupentixol and spiperone had no effect on VIP-modulated forskolin-stimulated adenylate cyclase activity. These results suggest that the neuromodulatory effect of VIP is mediated by a N_s distinct from those involved in several adenylate cyclase pools sensitive to stimulation by dopamine and VIP in the rat striatum.     

Secretin and VIP-stimulated adenylate cyclase from rat heart

Cardiac adenylate cyclase activity was normal in 3 weeks-old spontaneously hypertensive rats of the Wistar-Okamoto substrain. The hormone-sensitive adenylate cyclase activity was reduced in 10 weeks-old or older animals, and secretin- and VIP-activations were definitely more impaired (by 64% and 69%, respectively) than isoproterenol- and glucagonactivation (17% and 22%, respectively). By contrast, the fluoride- and p[NH]ppG-stimulations of the enzyme were unaffected. These alterations in the adenylate cyclase system coupled to secretin and VIP appeared specific to the heart as the isolated pancreatic acinar cells from spontaneously hypertensive animals responded normally to secretin, as a liver particulate fraction responded normally to secretin and VIP, and both brain synaptic membranes and a particulate fraction of anterior pituitary to VIP.Abbreviations VIP vasoactive intestinal peptide - cyclic AMP cyclic adenosine 35-monophosphate - p[NH]ppG guanosine 5-(, -imido)triphosphate - EGTA ethylene-glycol-bis-(2-amino-ether)-N,N,N,N-tetraacetic acid - IBMX 3-isobutyl-1-methylxanthine     

Distribution of vasoactive intestinal peptide-sensitive adenylate cyclase activity along the rabbit nephron

The effect of vasoactive intestinal peptide (VIP) upon adenylate cyclase (AC) activity has been determined in defined microdissected renal tubules isolated from collagenase-treated rabbit kidneys. In the presence of 10 M GTP, 1 M VIP gave marked stimulations of AC over basal values in the bright portion of the distal convoluted tubule (DCTb) (10.1-fold), and in the collecting tubule isolated from the inner stripe of the outer medulla (OMCTi, 7.8-fold). Less pronounced effects of VIP were found in the medullary collecting tubule isolated from the outer stripe (2.5-fold) and in the granular portion of the distal convoluted tubule (2.0-fold). VIP stimulation of AC activity in these segments amounted to 25 to 40% of the effect elicited by other agonists (arginine vasopressin, calcitonin or parathyroid hormone) in their respective target segments. A low response to VIP was observed in the cortical thick ascending limb (1.8-fold) which represented less than 5% of the calcitonin-stimulated AC activity. In the thin descending limb VIP produced a slight and variable stimulation of AC. VIP was without effect upon AC in the convoluted and straight portions of the proximal tubule, the medullary thick ascending limb and the cortical collecting tubule. Halfmaximal stimulation of AC by VIP was observed at 26±10 nM (n=3) in OMCTi and at 19 nM (n=2) in DCTb. Related peptides glucagon, secretin and PHI gave lower stimulations of AC compared to VIP in OMCTi. Conversely for rat OMCTi, under identical conditions, glucagon was much more effective than VIP.     

大鼠内毒素性发热时不同脑区cAMP含量和腺苷酸环化酶活性的变化

给大鼠腹腔注射大肠杆菌内毒素(endotoxin, ET)复制发热模型,观察大鼠ET性发热时不同脑区组织cAMP含量和腺苷酸环化酶(adenylate cyclase, AC)活性的变化。结果发现:大鼠在发热高峰时与对照组比较,丘脑下部cAMP含量明显增加(P<0.01),并与体温变化呈正相关关系(r=0.827);丘脑下部AC活性也显著增强(P<0.001),也与体温变化呈正相关关系(r=0.774)。脑干AC活性显著增强(P<0.05),但与体温变化无正相关关系(r=0.203),cAMP含量也无明显变化。脑皮质cAMP含量和AC活性均无明显变化。以上结果显示:ET可能是通过共同信息物质——内生致热原(EP),以一定方式作用于丘脑下部视前区神经元的细胞膜内AC,使其活化作用于ATP,使ATP分解生成cAMP,从而使局部cAMP含量增加,再通过某种方式使体温调定点上移,导致体温升高。     

Renal refractoriness to PTH in experimental Fanconi syndrome: Evidence for intact adenylate cyclase activation

Previous studies demonstrated renal refractoriness to parathyroid hormone (PTH) in maleate-induced Fanconi syndrome (FS) in rats.In membrane fraction of renal cortex of parathyroidectomized (PTX) rats with FS, PTH increased adenylate cyclase (AC) activity from a basal value of 7.4±0.6 (x±SE) to 16.8±3.5 pmoles cAMP/mg prot/min (P<0.01), similar stimulation by PTH was observed in control incubation with normal kidneys. In membrane fraction of renal cortex of PTX rats incubated with maleate, PTH increased AC from 5.7±0.42 to 10.7±1.08 (P<0.01) similar to control incubation without maleate.In cortical slices from PTX rates with FS incubated in vitro, PTH increased cAMP content only from 4.0±0.21 to 5.27±0.27 pmole cAMP/mg prot (P<0.005), while in slices from the control group the increment by PTH was from 3.9±0.43 to 10.7±0.93 pmoles cAMP/mg prot (P<0.001). For the difference in the increment between the control and FS group,P<0.001.In cortical tissue of PTX rats with FS, PTH injection failed to increase cAMP content: basal value 7.4±0.8 and with PTH 9.2±0.8 pmole cAMP/mg prot (P NS), as compared with controls: basal value 9.5±0.5 and with PTH 23.8±1.6 (P<0.001). ATP content of cortical slices fell from a control value of 2.3±0.18 in PTX rats to 1.0±0.14 nmol/mg prot in PTX rats with FS.These results show, (1) normal response to PTH stimulation of cortical AC from rats with FS, and (2) failure of PTH to enhance formation of cAMP in renal cortical tissue from rats with FS. These findings are consistent with an intact PTH receptor/AC system in maleate induced FS, but inability to increase cAMP production probably because of comprized cellular metabolism causing reduced ATP content.     

D2 dopamine receptor mRNA distribution in cholinergic and somatostatinergic cells of the rat caudate-putamen and nucleus accumbens

An in situ hybridization procedure that identifies cells expressing D2 dopamine receptor mRNA was combined in double-labelling studies with immunohistochemical procedures that identify cells expressing either choline acetyltransferase (ChAT) or somatostatin. D2 receptor mRNA was detected in almost all of the ChAT positive caudate-putamen cells, approximately half of the ChAT positive nucleus accumbens cells and none of the somatostatin-positive cells in either brain region.     

Defective leukocyte adenylate cyclase function in hypokalemia

Summary Patients with hypokalemia due to Bartter's syndrome show an increase in adrenergic nervous system function with significantly elevated plasma norepinephrine excretion. Prolonged exposure to neurotransmitters or hormones is known to lead to a down-regulation of target-cell responsiveness. We measured cyclic AMP generation by leukocytes in response to the -adrenergic agonist isoproterenol and to prostaglandin E₁ (PGE₁) in six patients with Bartter's syndrome. As compared to normal controls, the response of cyclic AMP production by leukocytes to stimulation by 1-isoproterenol or PGE₁ was significantly decreased. These results indicate that in Bartter's syndrome and probably in other diseases involving hypokalemia isoproterenol- and PGE₁-sensitive adenylate cyclase activities of leukocytes are reduced. Because the effect of PGE₁ on adenylate cyclase is not mediated through the specific -adrenoceptor, it is possible that a defect in receptor-adenylate cyclase coupling or a more distal post-receptor defect is responsible for the reduction in cyclic AMP production.Abbreviations cyclic AMP adenosine 3:5 - cyclic monophosphate Portions of this work have been presented at the Annual Meeting of the American Society for Clinical Investigation, April 29 to May 2, 1983, Washington, DC (Clin Res 1983; 31:471A)     

Characterization of a Mycobacterium tuberculosis mutant deficient in pH-sensing adenylate cyclase Rv1264

Mycobacterium tuberculosis open reading frame Rv1264 encodes an adenylate cyclase that exhibits its highest enzymatic activity at an acidic pH of 6.0. This is the pH M. tuberculosis encounters in the phagosome. Consequently Rv1264 has been suggested to sense the phagosomal milieu resulting in adaption of M. tuberculosis to its intracellular niche. A targeted knock-out mutant deficient in Rv1264, however, exhibits wild-type virulence.     

Effect of glucagon on adenylate cyclase activity and acid production of isolated human parietal cells

Summary The direct effect of glucagon on human parietal cell function in vitro was tested by measuring adenylate cyclase (AC) activity and H⁺ production in homogenates of human gastric mucosa obtained during surgery or at biopsy. Cells isolated from mucosa obtained during surgery showed an increase in AC with histamine and glucagon. In parietal cell enriched fractions (75%) glucagon and histamine stimulated AC much more effectively than in parietal cell depleted fractions (15% and 7%). In contrast, glucagon did not affect basal or histamine stimulated¹⁴C amino pyrine uptake.In homogenates of mucosal biopsy specimens 2×10^–7 mol/l glucagon enhanced AC activity by 76% (corpus) and 20% (antrum). In the same homogenates 10^–4 mol/l histamine caused a stimulation by 161% (corpus) and 38% (antrum). In fundic biopsy specimens glucagon displayed a biphasic concentration response curve with an increase at 10^–10 mol/l (46% above basal AC activity) and a maximum at 2×10^–7 mol/l (97%). Histamine elicited the maximal response (192%) at 10^–3 mol/l. Increasing histamine and glucagon concentrations caused additive stimulation of AC. Ranitidine did not change AC in response to glucagon but abolished the effect of histamine. Data suggest that the glucagon action is mediated by separate (glucagon?) receptors. As H⁺ production was not affected by glucagon, the coexistence of two AC systems in the human parietal cell is postulated: One that is activated via histamine H₂-receptors and which stimulates H⁺ production; another that is activated by glucagon and is directed towards other, possibly metabolic effects.Abbreviations AC Adenylate cyclase - cAMP Cyclic adenosine monophosphate - ATP Adenosine triphosphate Supported by grants from the Deutsche Forschungsgemeinschaft Mi 170/2-2Dedicated to Prof. Dr. F. Krück on the occasion of his 65th birthday     

Effects of reserpine treatment on β-adrenergic/adenylate cyclase modulated secretion and resynthesis by the rat submandibular gland

Summary Chronic reserpine (adrenergic blocking) treatment causes a marked accumulation of secretory protein in the rat submandibular gland (SMG) but discharge of this material is delayed in response to isoproterenol stimulation. The purposes of this study were to investigate the effects of chronic reserpine treatment on 1) the number of -adrenergic receptors, 2) the sensitivity of cell-surface-associated adenylate cyclase to various concentrations of isoproterenol, and 3) to correlate these data to morphologic studies of the secretion and resynthesis phases of the isoproterenol-induced secretory cycle in the rat SMG. Animals were injected with reserpine (0.5 g/g b.w.) for 6 days. Plasma membrane fractions were prepared. The adenylate cyclase response to a series of isoproterenol concentrations, and the number of -adrenergic receptors ([³H]-alprenolol binding) were determined. Other animals were given a single dose of isoproterenol (0.8 mg/100 g b.w.) and the SMG was examined by light and electron microscopy at various times (30 min to 24 h) after treatment. Chronic reserpine treatment leads to a 2.5-fold increase in SMG -adrenergic binding sites and a 50-fold increase in adenylate cyclase sensitivity to IPR stimulation when compared to controls. However, secretion and resynthesis of secretory product in response to IPR stimulation was greatly delayed in reserpinized rats.A preliminary report of this data has been previously presented: Cutler, L.S. et al. (1979) J. Dent. Res. 58A:380     

Expression of dopamine D2 receptor and choline acetyltransferase mRNA in the dopamine deafferented rat caudate-putamen

Summary In situ hybridization was used to study dopamine D2 receptor (D2R) and choline acetyltransferase (ChAT) mRNA expression in neurons of the rat forebrain, both on control animals and after a unilateral 6-hydroxydopamine (6-OHDA) lesion of midbrain dopamine neurons. D2R mRNA expressing neurons were seen in regions which are known to be heavily innervated by midbrain dopamine fibers such as caudate-putamen, nucleus accumbens and olfactory tubercle. ChAT mRNA expressing neurons were seen in caudate-putamen, nucleus accumbens and septal regions including vertical limb of the diagonal band. In caudate-putamen, approximately 55% of the medium sized neurons, which is the predominating neuronal cell-size in this region, were specifically labeled with the D2R probe. In addition, approximately 95% of the large size neurons in caudate-putamen were specifically labeled with both the D2R and ChAT probes, suggesting that most cholinergic neurons in the caudate-putamen express D2R mRNA. After a unilateral lesion of midbrain dopamine neurons, no change in the level of either D2R or ChAT mRNA were seen in the large size intrinsic cholinergic neurons in caudate-putamen. Similarily, no evidence was obtained for altered levels of D2R mRNA in medium size neurons in medial caudate-putamen, or nucleus accumbens. However, an increase in the number of medium size neurons expressing D2R mRNA was observed in the lateral part of the dopamine deafferented caudateputamen. Thus, it appears that midbrain dopamine deafferentation causes an increase in D2R mRNA expression in a subpopulation of medium size neurons in the lateral caudate-putamen.     

Characterization of the VIP- and secretin-stimulated adenylate cyclase system from human lung

The response of a crude particulate adenylate cyclase preparation from surgically removed human lung to guanine nucleotides, sodium fluoride, -adrenergic agonists, prostaglandins, vasoactive intestinal peptide (VIP), secretin, and [Val⁵]secretin was investigated. The enzyme activity increased 5, 10, and 9-fold, respectively, with GTP, Gpp(NH)p, and sodium fluoride. This activity was stimulated (in the presence as well as in the absence of added GTP) byd,l-isoproterenol,l-epinephrine andl-norepinephrine, the relative potency of these agonists being compatible with the existence of -adrenoceptors of the -adrenoceptors of the ₂ subtype. Prostaglandins E₁ and E₂, but not PGF₁ and PGF₂, stimulated the enzyme, PGE₁ being at least 10 times more potent than PGE₂. The biphasic pattern of stimulation of the same adenylate cyclase activity by VIP suggested the presence of high- and low-affinity VIP receptors coupled to the enzyme. This stimulation by VIP was not inhibited by secretin-(7–27). The stimulation of adenylate cyclase by secretin and [Val⁵]secretin was also biphasic, suggesting the coexistence of high- and low-affinity secretin receptors. Secretin-(7–27) was able to inhibit completely the secretin stimulation acting through high-affinity secretin receptors but exerted no effect on the stimulation operating through low-affinity secretin receptors, which might indicate that the latter receptors were in fact VIP-preferring receptors. [Val⁵]secretin was also used to differentiate these peptide receptors, since its properties were more VIP-like than those of secretin.Abbreviations VIP vasoactive intestinal peptide - Sn secretin - cyclic AMP cyclic adenosine 3:5-monophosphate - Gpp(NH)p guanosine 5-O-(2-3-imido)triphosphate - EGTA ethylene glycol bis (2-aminoethyl ether)-N,N,N,N-tetraacetic acid     

Abnormal activity of adenylate cyclase in the Drosophila memory mutant rutabaga

The activity of adenylate cyclase in homogenates prepared from the Drosophila memory mutant rutabaga is lower than normal. The effect is most pronounced in washed membranes prepared from the abdomen, in which the enzyme displays both a lower Vmax and a higher Km. Analysis of the effect of divalent cations suggests a lesion in the responsiveness of the enzyme to Mg2+. Studies of adenylate cyclase in rutabaga may potentially pinpoint a subpopulation of the enzyme which may prove relevant to molecular mechanisms underlying conditioning.     

Identification of unmyelinated axons in fractions from caudate-putamen in the rat

Summary Previous studies have indicated that fractions of unmyelinated axons can be obtained from cerebellum, whole brain or forebrain. It seemed likely that unmyelinated axons break into segments upon homogenization and that any circumscribed region of brain containing abundant unmyelinated axons could be found to yield axonal fractions. Caudate-putamen material was used to test this hypothesis. Fractions analogous to cerebellum were obtained from sucrose gradients and were found to yield abundant axonal profiles which could be correlated with the appearance of unmyelinated axons after sucrose immersion in situ. We conclude that fractions of unmyelinated axons can likely be obtained from any anatomical region rich in them and can be used upon purification for obtaining chemical data from different axonal types.Supported in part by NIH Grant 2-RO-1 NS-09745.     

Impairment of Hormone-Stimulated cardiac adenylate cyclase activity in the genetically obese (fa/fa) Zucker rat

The age-related development of the capacity of the cardiac adenylate cyclase system to be stimulated with secretin, vasoactive intestinal peptide (VIP), glucagon, the β-adrenergic agonist isoproterenol, Gpp(NH)p, and NaF was compared in obese (fa/fa) Zucker rats and their lean (FA/?) littermates. The obese (fa/fa) Zucker rats tested developed postweaning obesity associated with marked hypertriglyceridemia, mild hyperglycemia, and hyperinsulinism. At 4 weeks, there was already a 57% reduction in secretin-VIP-stimulated adenylate cyclase activity in fa/fa rats. At 12 weeks, the secretin-VIP-stimulation was reduced by 77%, and glucagon-and isoproterenol-stimulations by 16–21%. At 45 weeks, secretin-VIP-stimulation was reduced by 91%, glucagon- and isoproterenol stimulations by 34–42%, and Gpp(NH)p- and NaF-stimulations by 16–23%. The reductions of isoproterenol-, Gpp(NH)p-, and NaF-stimulations were totally or partially reversed in 30-week old fa/fa animals submitted for 5 weeks to severe food restriction that almost normalized the altered blood parameters. In sharp contrast, food restriction imposed a further decrease in secretin-VIP- and glucagon-stimulated adenylate cyclase activities. This pattern of impaired secretin-VIP-stimulated adenylate cyclase activity appeared limited to cardiac membranes in obese animals as the responses of liver, brain and anterior pituitary adenylate cyclase activities to secretin and/or VIP were unaltered. These results suggest that secretin-VIP receptors coupled to adenylate cyclase were rapidly and specifically altered in the heart of fa/fa Zucker rats.     

Inhibition of adenylate cyclase attenuates muscarinic Ca signaling by a PKA-independent mechanism in rat carotid body Type I cells

Carotid body (CB) Type I cells respond to hypoxia by releasing excitatory and inhibitory neurotransmitters. This mechanism leads to increased firing of the carotid sinus nerve (CSN) which alters breathing to maintain blood gases within the physiological range. Acetylcholine targets both muscarinic and nicotinic receptors in the rat CB, acting postsynaptically on CSN and presynaptically on Type I cells. Muscarinic Ca²⁺ signaling is inhibited by the activation of G_i-coupled receptors including histamine H3 receptors. Here inhibition of adenylate cyclase with SQ22536 mimicked H3 receptor activation. Using Ca²⁺ imaging techniques it was observed that inhibition of muscarinic Ca²⁺ signaling was independent of protein kinase A (PKA) as PKA inhibitors H89 and KT5720 were without effect on the muscarinic Ca²⁺ response. By contrast the Epac (exchange protein activated by cAMP) inhibitor brefeldin A inhibited muscarinic Ca²⁺ signaling whereas the Epac activator 8-pCPT-2′-O-Me-cAMP-AM potentiated Ca²⁺ signaling.