A defective conditioned behavior and a defective adenylate cyclase in theDrosophila mutantrutabaga

Summary TheDrosophila X-linked mutantrutabaga (Duerr and Quinn 1982) fails to perform normally in olfactory conditioning paradigms, in spite of being able to sense odorants and shock (Figs. 1–3).rut is capable of forming an association between shock and odorant, but memory decays rapidly; the memory of the mutant following intensive training resembles that of normal flies following very brief training (Fig. 4).rut flies also display in vitro a defective adenylate cyclase activity (Fig. 6). The enzyme in the mutant is responsive to stimulation by a putative neurotransmitter and by a guanyl nucleotide (Fig. 8) but the activity is lower than normal even in the presence of forskolin (Fig. 8) and MnATP (Fig. 9), suggesting that the lesion is closely associated with the function of the catalytic subunit.rut/ + heterozygotes are semi-recessive with regard to both the behavioral defect and the biochemical defect (Figs. 5, 7). The behavioral and the biochemical lesions detected inrut flies are discussed in light of current molecular models of learning.     

Ca2+-dependent regulation of calmodulin binding and adenylate cyclase activation in bovine cerebellar membranes

The binding parameters of ¹²⁵I-labeled calmodulin to bovine cerebellar membranes have been determined and correlted with the activation of adenylate cyclase by calmodulin. In the presence of saturating levels of free Ca²⁺, calmodulin binds to a finite number of specific membrane sites with a dissociation constant (K_d) of 1.2 nM. Furthermore, Scatchard analysis reveals a second population of binding sites with a 100-fold lower affinity for calmodulin. The Ca²⁺-dependence of calmodulin binding and of adenylate cyclase activation varies with the amount of calmodulin present, as can be infered from the model of sequential equilibrium reactions which describes the activation of calmodulin-dependent enzymes. On the basis of this model, a quantitative analysis of the effect of free Ca²⁺ and of free calmodulin concentration on both binding and activation of adenylate cyclase was carried out. This analysis shows that both processes take place only when calmodulin is complexed with at least three Ca²⁺ atoms. The concentration of the active calmodulin ·Ca²⁺ species required for half-maximal activation of adenylate cyclase is very similar to the K_d of the high affinity binding sites on brain membranes. A Hill coefficient of approx. 1 was found for both processes indicating an absence of cooperativity. Phenothiazines and thioxanthenes antipsychotic agents inhibit calmodulin binding to membranes and calmodulin-dependent activation of adenylate cyclase with a similar order of potency. These results suggest that the Ca²⁺-dependent binding of calmodulin to specific high affinity sites on brain membranes regulates the activation of adenylate cyclase by calmodulin.     

The interaction of catecholamines,Ca2+ and adenylate cyclase in the intact turkey erythrocyte

Epinephrine stimulated adenylate cyclase in turkey erythrocyte ghosts is inhibited by calcium. The inhibition of adenylate cyclase is not apparent when intact erythrocytes are incubated with calcium and epinephrine. However, in the presence of the specific cation ionophore A₂₃₁₈₇ and 5 mm Ca²⁺, a 90% inhibition of epinephrine stimulated 3′,5′-adenosine monophosphate formation is found. The effect of catecholamines on calcium transport in the intact turkey erythrocyte was studied. Epinephrine causes a small but significant increase in Ca²⁺ efflux. This effect is inhibited by propranolol. No effect of epinephrine on Ca²⁺ uptake was observed. However, a 22% increase in Ca²⁺ uptake in the presence of propranolol could be detected. The propranolol effect was found to possess high statistical significance (p < .001). The absence of an epinephrine effect on influx probably reflects the presence of endogenous catecholamines in the control samples.It is proposed that the activation of adenylate cyclase by catecholamines occurs in two phases. The first phase is the increase of net Ca²⁺ efflux from a crucial Ca²⁺ pool, thus removing Ca²⁺ from its inhibitory sites on the adenylate cyclase complex. The second phase is the activation of the deinhibited adenylate cyclase by the hormone.     

Adenylate cyclase in sea anemone implication for chemoreception

Adenylate cyclase of the sea anemoneAnthopleura elegantissima was found to be associated with the heavy particulate fraction of the cell and to be activated by NaF and 2-mercaptoethanol. Reduced glutathione, which elicits the ciliary swallowing response during feeding, also activated adenylate cyclase in particles from the oral disc and pharynx. The GSH effect was dependent on homogenization procedure, whereas the NaF and 2-mercaptoethanol activation was not. The activation of adenylate cyclase from the oral disc and pharynx by GSH was correlated with increased Ca²⁺ binding to the particulate fraction. When activation by GSH was abolished by mechanical homogenization, no increasea in Ca²⁺ binding was observed in the presence of GSH. It is suggested that chemoreception for the swallowing response of this organism is mediated by cyclic AMP control of Ca²⁺ distribution in the cell.     

Calcium regulation of guanine nucleotide activation of hepatic adenylate cyclase

Pretreatment of isolated rat liver plasma membranes by washing with NaHCO₃ buffer or by exposure to the chelator ethyleneglycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) with or without the ionophore A23187, produced a decrease in the sensitivity of adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) to subsequent stimulation by NaF or guanosine 5′-(β-γ-imino)triphosphate (GPP(NH)P). Sensitivity to activation by the nucleotide could be restored by addition of the lyophilized and ashed wash or by addition of Ca²⁺, Mg²⁺ or Mn²⁺. The factor extracted from the membranes by these various treatments which was responsible for loss of stimulation was identified as Ca²⁺. Determination of the metal ion content of isolated membranes by atomic absorption spectrometry indicated that Ca²⁺ was the only divalent cation present in sufficient concentration to support persistent activation by either NaF or GPP(NH)P.Pretreatment of liver plasma membranes with trifluoperazine, which inhibits the action of Ca²⁺-dependent regulator protein in other enzyme systems, reduced GPP(NH)P activation of adenylate cyclase and caused marked depletion of membrane Ca²⁺. The effects of low concentrations (less than 100 μM) of the phenothiazine could be reversed totally by Ca²⁺ and partly by regulator protein. At higher concentrations of trifluoperazine, slight restoration of enzyme activation was seen with either agent. The hypothesis is presented that Ca⁺ interacts with the nucleotide (GTP or GDP) regulatory site(s) of the adenylate cyclase. This interaction may be regulator-protein-dependent and may be important in determining the sensitivity of the enzyme to nucleotide activation in vivo.     

Modulation of dihydropyridine‐sensitive calcium channels in drosophila by a cAMP‐mediated pathway

Drosophila has proved to be a valuable system for studying the structure and function of ion channels. However, relatively little is known about the regulation of ion channels, particularly that of Ca²⁺ channels, in Drosophila. Physiological and pharmacological differences between invertebrate and mammalian L‐type Ca²⁺ channels raise questions on the extent of conservation of Ca²⁺ channel modulatory pathways. We have examined the role of cyclic adenosine monophosphate (cAMP) cascade in modulating the dihydropyridine (DHP)‐sensitive Ca²⁺ channels in the larval muscles of Drosophila, using mutations and drugs that disrupt specific steps in this pathway. The L‐type (DHP‐sensitive) Ca²⁺ channel current was increased in the dunce mutants, which have high cAMP concentration owing to cAMP‐specific phosphodiesterase (PDE) disruption. The current was decreased in the rutabaga mutants, where adenylyl cyclase (AC) activity is altered thereby decreasing the cAMP concentration. The dunce effect was mimicked by 8‐Br‐cAMP, a cAMP analog, and IBMX, a PDE inhibitor. The rutabaga effect was rescued by forskolin, an AC activator. H‐89, an inhibitor of protein kinase‐A (PKA), reduced the current and inhibited the effect of 8‐Br‐cAMP. The data suggest modulation of L‐type Ca²⁺ channels of Drosophila via a cAMP‐PKA mediated pathway. While there are differences in L‐type channels, as well as in components of cAMP cascade, between Drosophila and vertebrates, main features of the modulatory pathway have been conserved. The data also raise questions on the likely role of DHP‐sensitive Ca²⁺ channel modulation in synaptic plasticity, and learning and memory, processes disrupted by the dnc and the rut mutations. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 491–500, 1999     

Subcellular location of adenylate cyclase in rat cardiac muscle

Crude homogenates of rat cardiac muscle were fractionated in order to examine the subcellular location of adenylate cyclase in this tissue. The fractionation procedure employed differential centrifugation of homonized material, followed by collagenase treatment, centrifugation on a discontinuous sucrose density gradient and extraction with 1 M KCl. The particulate fraction obtained by this procedure contained a high specific activity and yield of adenylate cyclase, moderate levels of mitochondria and low levels of sarcoplasmic reticulum and contractile protein as judged by marker enzyme activities. Adenylate cyclase was purified 20-fold with a 33% yield from the crude homogenate, while mitochondrial, sarcoplasmic reticulum and contractile protein yields were 5, 0.4 and 0.7% respectively. The membrane fractions prepared in this manner were examined by sodium dodecyl sulfate · gel electrophoresis.Adenylate cyclase copurified with ouabain-sensitive (Na⁺ + K⁺)-ATPase, a plasma membrane marker enzyme, and not with Ca²⁺-accumulating activity, which is associated with the sarcoplasmic reticulum. The distribution of marker enzyme activities indicates that heart adenylate cyclase is not located in the sarcoplasmic reticulum but is localized predominantly, if not exclusively, in the plasma membrane.     

Renal parathyroid hormone-dependent adenylate cyclase activity after repletion of vitamin D-deficient rats with vitamin D-2

Rats fed a diet deficient in both vitamin D and Ca²⁺ exhibited a greater depression of the renal parathyroid hormone (PTH)-dependent adenylate cyclase than was observed in rats fed diets deficient in either vitamin D or calcium. Total serum Ca²⁺ was decreased from a control level of 11.2 mg/dl to 8.5 mg/dl in rats fed the diet deficient in calcium alone, and to 5.4 mg/dl in rats fed the diet deficient in vitamin D. Serum calcium was decreased further to 4.3 mg/dl in rats fed the diet deficient in both vitamin D and Ca²⁺. Serum immunoreactive PTH was significantly elevated over control levels when rats were fed the test diets; however, there were no significant differences between the elevated levels in the three experimental groups. Repletion of rats deficient in vitamin D only with a single oral dose of 3200 I.U. vitamin D-2 resulted in restoration of serum calcium to normal levels, a return of serum PTH to the control state, and an associated increase in PTH-dependent adenylate cyclase activity to the control level by 72 h. Repletion of rats deficient in both vitamin D and Ca²⁺ with the same dose of vitamin D-2 raised serum Ca²⁺ to 7.2 mg/dl by 72 h, but did not cause a reduction in circulating PTH, nor did it result in any significant improvement in the responsiveness of the membrane adenylate cyclase to PTH. These results suggest that elevated PTH is a factor in the down regulation of the PTH-dependent adenylate cyclase, but do not rule out a role for calcium as a regulatory factor.     

Demonstration and Characterization of Opiate Inhibition of the Striatal Adenylate Cyclase

Abstract: The conditions in which Leu⁵-enkephalin inhibition of striatal adenylate cyclase was observed were defined. It was determined that enkephalin inhibition was dependent on GTP. The apparent K_m for GTP in opiate inhibition was determined to be 0.5 and 2 μM when 0.1 mM- and 0.5 mM-ATP were used as substrate. ITP, but not CTP or UTP, could substitute for GTP in the reaction. Though the addition of monovalent cations—Na⁺,K⁺, Li⁺, Cs⁺, and choline⁺—stimulated striatal adenylate cyclase activity, enkephalin inhibition of striatal adenylate cyclase did not require Na⁺ when theophylline was used as the phosphodiesterase inhibitor. Under optimal conditions, i.e., 20 μM-GTP and 100 mM-Na⁺, Leu⁵-enkephalin inhibited the striatal adenylate cyclase activity by 23–27%. When the enkephalin regulation of the cyclase activity was further characterized, it was observed that Leu⁵-enkephalin inhibited the rate of the enzymatic reaction. Kinetic analysis revealed that the opioid peptide decreases V_max values but not the K_m values for the substrates Mg²⁺ and Mg-ATP. Agents such as MnCl₂, NaF, and guanyl-5′-ylimido-diphosphate, which directly activated the adenylate cyclase, antagonized the opiate inhibition. Levorphanol and (–)naloxone were more potent than dextrorphan and (+)naloxone in inhibiting adenylate cyclase and in reversing the enkephalin inhibition, respectively. There were differences in the potencies of various opiate peptides in their inhibition of striatal adenylate cyclase activity, with Met⁵- > Leu⁵-enkephalin > β-endorphin. The opiate receptor through which the enkephalin inhibition was observed is most likely δ in nature, since in the presence of either Na⁺ or K⁺, the magnitude of the alkaloid inhibition was reduced, whereas the peptide inhibition was either potentiated or not affected.     

Calcium ion effects on guanylate cyclase of brain.

Soluble guanylate cyclase activity of brain is stimulated by Ca²⁺ in the presence of low concentrations of Mn²⁺. Unlike Ca²⁺ stimulation of adenylate cyclase, the effect does not depend upon interaction of guanylate cyclase with a specific high-affinity Ca²⁺-binding protein. In the presence of Mg²⁺, Ca²⁺ inhibits soluble guanylate cyclase as well as the particulate enzyme. The concept that stimulation of brain cells results in increased cyclic GMP concentration secondary to Ca²⁺ influx merits additional critical study.     

A proper transmembrane Ca2+ gradient is essential for the stimulation of adenylate cyclase activity by stimulatory GTP-binding protein

Stimulatory GTP-binding Protein (Gs) and adenylate cyclase prepared from bovine brain cortices were co-reconstituted into asolectin vesicles with or without 1000-fold transmembrane Ca²⁺ gradient. The results showed that both basal activity and Gs-stimulated activity of adenylate cyclase were highest in proteoliposomes with a transmembrane Ca²⁺ gradient similar to physiological condition (1 M Ca²⁺ outside and 1 mM Ca²⁺ inside) and lowest when the transmembrane Ca²⁺ gradient was in the inverse direction. Such a difference could be diminished following dissipation of the transmembrane Ca²⁺ gradient by A23187. Comparable conformational changes of Gs in proteoliposomes were also observed when Gs was labeled with the fluorescence probe, acrylodan. These results may indicate that a proper transmembrane Ca²⁺ gradient is essential not only for higher adenylate cyclase activity but also for its stimulation by Gs.     

Role of phosphatidylinositol turnover in alpha1 and of adenylate cyclase inhibition in alpha2 effects of catecholamines

Ligand binding and pharmacological studies have indicated that alpha-adrenergic receptors can be divided into alpha₁ and alpha₂. We suggest that alpha₁ receptors mediate those metabolic effects of alpha catecholamines which involve phosphatidylinositol turnover and the release of bound intracellular Ca²⁺ as well as the entry of extracellular Ca²⁺. In contrast, alpha effects of catecholamines are due to non-specific inhibition of adenylate cyclase through a mechanism independent of Ca²⁺. A similar classification for the effects of both histamine and serotonin suggests that they have separate type 1 or alpha receptors for Ca²⁺ dynamics which are different from type 2 or beta receptors which regulate adenylate cyclase.There is a significant correlation between hormone effects on phosphatidylinositol turnover and elevation of intracellular Ca²⁺. The available data suggest that the turnover of membrane-bound phosphatidylinositol is involved in Ca²⁺ gating in rat hepatocytes, rat and hamster adipocytes and blowfly salivary glands. In hamster adipocytes adenylate cyclase activity is also inhibited by alpha₂ catecholamines through a Ca²⁺ independent mechanism.     

Divalent cation and lipid-protein interactions of biomembranes

Divalent cations play an important role in the functions of biomembranes. This review deals with three topics: (1) Mg²⁺-mediated change in physical state of phospholipid induces conformation and activity change of reconstituted mitochondrial H⁺-ATPase, (2) a proper transmembrane Ca²⁺ gradient is essential for the higher enzymatic activity of adenylate cyclase, and (3) role of transmembrane Ca²⁺ gradient in the modulation of reconstituted sarcoplasmic reticulm Ca²⁺-ATPase activity.     

Biochemical characterization of histamine-sensitive adenylate cyclase in mammalian brain.

Certain biochemical characteristics of an adenylate cyclase that is activated by low concentrations of histamine (K_a, 8 μm) and that is present in cell-free preparations from the dorsal hippocampus of guinea pig brain have been studied. Histamine increased the maximal reaction velocity of adenylate cyclase without altering the K_m (0.18 mm) for its substrate, MgATP. Increasing concentrations of free Mg²⁺ stimulated enzymatic activity; the kinetic properties of this activation by Mg²⁺ suggest the existence of a Mg²⁺ allosteric site on the enzyme. Histamine increased the affinity of this apparent site for free Mg²⁺. Free ATP was a competitive inhibitor with respect to the MgATP substrate. The apparent potency of free ATP as an inhibitor increased in the presence of histamine. In the presence of Mg²⁺, low concentrations of Ca²⁺ markedly inhibited adenylate cyclase activity; half-maximal inhibition of both basal and histamine-stimulated enzyme activity occurred at 40 μm Ca²⁺. Other divalent cations, including Zn²⁺, Cu²⁺, and Cd²⁺, were also inhibitory. Of the divalent cations tested, only Co²⁺ and Mn²⁺ could replace Mg²⁺ in supporting histamine-stimulated adenylate cyclase activity. The nucleoside triphosphates GTP and ITP increased basal adenylate cyclase activity and markedly potentiated the stimulation by histamine. Preincubation of adenylate cyclase with 5′-guanylylimidodiphosphate dramatically increased enzyme activity; in this activated state, the adenylate cyclase was relatively refractory to further stimulation by histamine or F^?. The subcellular distribution of histamine-sensitive adenylate cyclase activity was studied in subfractions from guinea pig cerebral cortex. The highest total and specific activities were observed in those fractions enriched in nerve endings, while adenylate cyclase activity was not detectable in the brain cytosol fraction. A possible physiological role for this histamine-sensitive adenylate cyclase in neuronal function is discussed.     

Stimulation of brain adenylate cyclase activity by the undecapeptide substance P and its modulation by the calcium ion

Synthetic substance P stimulated adenylate cyclase activity in particulate preparations from rat and human brain.The concentration of substance P for half maximal stimulation in rat brain was 1.8 · 10⁻⁷ M.The stimulatory effect of substance P on the rat brain adenylate cyclase activity was 88% compared with 48% by noradrenalin, 163% by prostaglandin E₁ and 184% by prostaglandin E₂.Both the basal and substance P-stimulated adenylate cyclase activity in rat brain were inhibited by concentration of Ca²⁺ above 10⁻⁶ M.The chelating agent ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid at a concentration of 0.1 mM reduced the basal adenylate cyclase activity by 64% and eliminated the substance P-stimulated activity.The inhibition by ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid was completely reversed by increasing concentrations of Ca²⁺.     

MOUSE NEUROBLASTOMA CELL ADENYLATE CYCLASE: REGULATION BY 2-CHLOROADENOSINE, PROSTAGLANDIN E1 AND THE CATIONS Mg2+, Ca2+ AND Mn

—Some basic kinetic properties of adenylate cyclase in cell free preparations of mouse neuroblastoma were investigated. Production of cAMP from ATP by the enzyme requires the presence of either Mg²⁺ or Mn²⁺ in addition to ATP. In the presence of Mg²⁺, the K_m for ATP is 120 ± 15 μM and the interaction of ATP and adenylate cyclase appears to be non-cooperative (Hill coefficient of 1). Magnesium ion concentrations in excess of the ATP concentration cause stimulation although similar excess concentrations of Mn²⁺ cause inhibition. Prostaglandin E₁ and 2-chloroadenosine activate the enzyme. The K_m of the cyclase for 2-chloroadenosine is 6 μm . Activation by 2-chloroadenosine leads to an increase in V_max but does not effect the K_m for ATP. At a fixed ATP concentration, the extent of activation caused by prostaglandin E₁ and 2-chloroadenosine is inversely related to the Mg²⁺ concentration. Calcium ion causes inhibition of adenylate cyclase from 0.1 to 4mM with a K_i of 5 ± 10^?4m . Ca²⁺ interaction with the enzyme in the absence or presence of either 2-chloroadenosine or prostaglandin E₁ appears cooperative (i.e. Hill coefficients of ?2). Ca²⁺ inhibition is non-competitive with respect to either ATP or 2-chloroadenosine but is progressively diminished by increasing Mn²⁺ concentrations. Divalent cation effects and activation by 2-chloroadenosine and prostaglandin E₁ of the neuroblastoma adenylate cyclase are compared with ion effects and hormone activation of the enzyme obtained from non-neuronal tissue.     

Studies on plasma membranes. XXI. Inhibition of liver plasma membrane enzymes by tumour-promoting phorbolester, mitotic inhibitors and cytochalasin B

The tumour promotor tetradecanoyl phorbol acetate (TPA) inhibited the Mg²⁺-, Ca²⁺- and (Na⁺-K⁺)ATPases of rat-liver plasma membranes. A nonpromoting phorbolester derivative was without effect. Colchicine and/or vinblastine inhibited the (Na⁺-K⁺)ATPase, glucagon-stimulated adenylate cyclase, and cyclic adenosine-3, 5-monophosphate (c-AMP) phosphodiesterase, but were without significant effect on the Mg²⁺- or Ca²⁺-ATPase. Cytochalasin B inhibited the (Na⁺-K⁺)ATPase. The results furnish the first direct evidence that these drugs may interact with plasma membranes. The mechanism of the enzyme inhibitions is briefly discussed.     

Calcium and adenosine affect human sperm adenylate cyclase activity

The adenylate cyclase activity of human ejaculated spermatozoa in broken-cell preparations was investigated. In the presence of 5 mM metal cations and 0.1 mM ATP, the relative enzyme activity with Mn²⁺, Ca²⁺, Mg²⁺, Ba²⁺ was 1.00, 0.28, 0.22, and 0.03, respectively. Added Ca²⁺ appeared to activate the enzyme in the presence of Mn²⁺ or Mg²⁺. The human sperm adenylate cyclase was stimulated by ～ 2-fold by free Ca²⁺ (lmM) in the presence of Mg²⁺ (5 mM). If the GTP analogue, 5′-guanylyl imidophosphate (Gpp(NH)p) was added to the sperm homogenate in the presence of 200 μM ethylene-glycol-bis (β-aminoethylether) N,N′-tetraacetic acid (EGTA), the adenylate cyclase activity was increased by approximately 25%, but with the addition of 280 μM Ca²⁺ there was a decrease in enzyme activity. A similar response to low concentrations of Ca²⁺ was obtained after complementation of the sperm enzyme with the guanine nucleotide regulatory component from human erythrocytes, where the addition of 40 μM Gpp(NH)p, 200 μM EGTA, and Ca²⁺ (≤ 160 μM) stimulated the sperm enzyme ～ 3–4-fold, but the further addition of Ca²⁺ (280 μM, final) neutralized the stimulatory effect. The addition of adenosine, and the nucleotides 5′-AMP and 5′-ADP inhibited the enzyme, whereas guanine and 5′-GMP had no appreciable effect. Human follicular fluid and serum also had little direct effect on the sperm adenylate cyclase. These resuls suggest that Ca²⁺ might be an important physiological modulator of the human sperm adenylate cyclase.     

Calcium-dependent adenylate cyclase of pituitary tumor cells

Effects of Ca²⁺ and calmodulin on the adenylate cyclase activity of a prolactin and growth hormone-producing pituitary tumor cell strain (GH₃) were examined. The adenylate cyclase activity of homogenates was stimulated approx. 60% by submicromolar free Ca²⁺ concentrations and inhibited by higher (μM range) concentrations of the cation. A 2–3-fold stimulation of the activity in response to Ca²⁺ was observed at physiologic concentrations of KCl, with both the stimulatory and inhibitory responses occurring at respectively higher free Ca²⁺ concentrations. Calmodulin in incubations at low KCl concentrations increased the enzyme activity at all Ca²⁺ concentrations tested. In incubations conducted at physiologic KCl concentrations, both the inhibitory and stimulatory responses to Ca²⁺ were shifted by calmodulin to lower respective concentrations of the cation, without significant change occurring in the maximal rate of enzymic activity at optimal free Ca²⁺. Mg²⁺ concentrations in the incubation also influenced the Ca²⁺ concentration dependence of adenylate cyclase; at high Mg²⁺ more Ca²⁺ was required to obtain maximal activity. Trifluoperazine inhibited adenylate cyclase of GH₃ cells only in the presence of Ca²⁺; as Ca²⁺ concentrations in the assay were increased, higher drug concentrations were required to inhibit the enzyme. Ca²⁺ was also observed to reduce the extent of enzyme destabilization which occurred during pretreatments at warm temperatures. Vasoactive intestinal polypeptide and phorbol myristate acetate, which stimulate prolactin secretion in intact GH₃ cells, enhanced enzyme activity 4- and 2.5-fold, respectively, without added Ca²⁺. Increasing free Ca²⁺ concentrations reduced the enhancement by VIP and eliminated the stimulation by PMA.     

Changes in plasma membrane enzyme activities during liver regeneration in the rat

Changes in activities of plasma membrane enzymes during liver regeneration may be related to the maintenance of hepatic function or to the regulation of cell proliferation. Plasma membranes were isolated from rat livers at various times after partial hepatectomy, and the specific activities of alkaline phosphatase, (Na⁺ + K⁺)-ATPase, leucine aminopeptidase, 5′-nucleotidase, and adenylate cyclase (basal and with glucagon or epinephrine) were measured. Alkaline phosphatase and (Na⁺ + K⁺)-ATPase activity increased 3.6-fold and 2-fold respectively, during the first 48 h after partial hepatectomy. The time of onset and duration of change suggest that these increases in activity are involved in the maintenance of bile secretion. Decreases in leucine aminopeptidase activity at 48–108 h and in 5′-nucleotidase activity at 12–24 h were observed, which may be involved in the restoration of protein and accumulation of RNA. The basal activity of adenylate cyclase increased after partial hepatectomy. The response of adenylate cyclase to epinephrine showed a transitory increase between 36 and 108 h after surgery, while the response to glucagon was decreased by approximately 50% at all time points through 324 h after surgery. These changes in the hormone responsiveness of adenylate cyclase are similar to those previously observed in fetal and preneoplastic liver.