Molecular cloning and characterization of the human cardiac Na+/Ca2+ exchanger cDNA.

The Na+/Ca2+ exchanger plays important roles in Ca2+ handling in many excitable cells. In particular, the Na+/Ca2+ exchanger is expressed at high levels in the cardiac sarcolemma and is the dominant mechanism of Ca2+ extrusion from the cells. In addition, the exchanger has been suggested to play key roles in digitalis action and in postischemic reperfusion injury of cardiac myocytes. We report here the isolation and characterization of the cDNA encoding the human cardiac Na+/Ca2+ exchanger. Twelve overlapping clones corresponding to 5.6 kilobases of the exchanger cDNA sequence were isolated from 5 x 10(5) phage plaques screened. The sequence predicted a 973-amino acid polypeptide with a putative leader peptide, 11 potential membrane-spanning regions, and one large putative cytoplasmic loop between the fifth and sixth transmembrane helices. When RNA was synthesized in vitro from the cloned cDNA and injected into Xenopus oocytes, it induced expression of Na+/Ca2+ exchange activity at high levels, confirming that this clone encodes the functional Na+/Ca2+ exchanger. Southern blot analysis indicated that the cardiac exchanger gene exists as a single copy in the human genome, although existence of other related genes cannot be ruled out. Northern blot and S1 mapping analyses revealed that the cardiac type exchanger mRNA is expressed most abundantly in the heart and next in the brain. The cardiac-type exchanger mRNA was also expressed in the retina and in skeletal and smooth muscles at very low levels. The levels of mRNA encoding the exchanger were significantly lower in fetal hearts than in adult hearts but were unchanged in the myocardium from patients with end-stage heart failure.     

Regulation of phosducin phosphorylation in retinal rods by Ca2+/calmodulin-dependent adenylyl cyclase.

The phosphoprotein phosducin (Pd) regulates many guanine nucleotide binding protein (G protein)-linked signaling pathways. In visual signal transduction, unphosphorylated Pd blocks the interaction of light-activated rhodopsin with its G protein (Gt) by binding to the beta gamma subunits of Gt and preventing their association with the Gt alpha subunit. When Pd is phosphorylated by cAMP-dependent protein kinase, it no longer inhibits Gt subunit interactions. Thus, factors that determine the phosphorylation state of Pd in rod outer segments are important in controlling the number of Gts available for activation by rhodopsin. The cyclic nucleotide dependencies of the rate of Pd phosphorylation by endogenous cAMP-dependent protein kinase suggest that cAMP, and not cGMP, controls Pd phosphorylation. The synthesis of cAMP by adenylyl cyclase in rod outer segment preparations was found to be dependent on Ca2+ and calmodulin. The Ca2+ dependence was within the physiological range of Ca2+ concentrations in rods (K1/2 = 230 +/- 9 nM) and was highly cooperative (n app = 3.6 +/- 0.5). Through its effect on adenylyl cyclase and cAMP-dependent protein kinase, physiologically high Ca2+ (1100 nM) was found to increase the rate of Pd phosphorylation 3-fold compared to the rate of phosphorylation at physiologically low Ca2+ (8 nM). No evidence for Pd phosphorylation by other (Ca2+)-dependent kinases was found. These results suggest that Ca2+ can regulate the light response at the level of Gt activation through its effect on the phosphorylation state of Pd.     

Molecular cloning, characterization, and functional expression of rat oxidosqualene cyclase cDNA.

A cDNA encoding rat oxidosqualene lanosterol-cyclase [lanosterol synthase; (S)-2,3-epoxysqualene mutase (cyclizing, lanosterol-forming), EC 5.4.99.7] was cloned and sequenced by a combination of PCR amplification, using primers based on internal amino acid sequence of the purified enzyme, and cDNA library screening by oligonucleotide hybridization. An open reading frame of 2199 bp encodes a M(r) 83,321 protein with 733 amino acids. The deduced amino acid sequence of the rat enzyme showed significant homology to the known oxidosqualene cyclases (OSCs) from yeast and plant (39-44% identity) and still retained 17-26% identity to two bacterial squalene cyclases (EC 5.4.99.-). Like other cyclases, the rat enzyme is rich in aromatic amino acids and contains five so-called QW motifs, highly conserved regions with a repetitive beta-strand turn motif. The binding site sequence for the 29-methylidene-2,3-oxidosqualene (29-MOS), a mechanism-based irreversible inhibitor specific for the vertebrate cyclase, is well-conserved in all known OSCs. The hydropathy plot revealed a rather hydrophilic N-terminal region and the absence of a hydrophobic signal peptide. Unexpectedly, this microsomal membrane-associated enzyme showed no clearly delineated transmembrane domain. A full-length cDNA was constructed and subcloned into a pYEUra3 plasmid, selected in Escherichia coli cells, and used to transform the OSC-deficient uracil-auxotrophic SGL9 strain of Saccharomyces cerevisiae. The recombinant rat OSC expressed was efficiently labeled by the mechanism-based inhibitor [3H]29-MOS.     

Cloning and expression of a Ca(2+)-inhibitable adenylyl cyclase from NCB-20 cells.

A cDNA that encodes an adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] has been cloned from NCB-20 cells, in which adenylyl cyclase activity is inhibited by Ca2+ at physiological concentrations. The cDNA clone (5.8 kilobases) was isolated by polymerase chain reaction (PCR) using degenerate primers designed by comparison of three adenylyl cyclase sequences (types I, II, and III) and subsequent library screening. Northern analysis revealed expression of mRNA (6.1 kilobases) corresponding to this cDNA in cardiac tissue, which is a prominent source of Ca(2+)-inhibitable adenylyl cyclase. The clone encodes a protein of 1165 amino acids, whose hydrophilicity profile was very similar to those of other mammalian adenylyl cyclases that have recently been cloned. A noticeable difference between this protein and other adenylyl cyclases was a lengthy aminoterminal region before the first transmembrane span. Transient expression of this cDNA in the human embryonic kidney cell line 293 revealed a 3-fold increase in cAMP production in response to forskolin compared with control transfected cells. In purified plasma membranes from transfected cells, increased adenylyl cyclase activity was also detected, which was susceptible to inhibition by submicromolar Ca2+. Thus, this adenylyl cyclase seems to represent the Ca(2+)-inhibitable form that is encountered in NCB-20 cells, cardiac tissue, and elsewhere. Its identification should permit a determination of the structural features that determine the mode of regulation of adenylyl cyclase by Ca2+.     

Molecular cloning and characterization of SCaMPER, a sphingolipid Ca2+ release-mediating protein from endoplasmic reticulum.

Release of Ca2+ stored in endoplasmic reticulum is a ubiquitous mechanism involved in cellular signal transduction, proliferation, and apoptosis. Recently, sphingolipid metabolites have been recognized as mediators of intracellular Ca2+ release, through their action at a previously undescribed intracellular Ca2+ channel. Here we describe the molecular cloning and characterization of a protein that causes the expression of sphingosyl-phosphocholine-mediated Ca2+ release when its complementary RNA is injected into Xenopus oocytes. SCaMPER (for sphingolipid Ca2+ release-mediating protein of endoplasmic reticulum) is an 181 amino acid protein with two putative membrane-spanning domains. SCaMPER is incorporated into microsomes upon expression in SO cells or after translation in vitro. It mediates Ca2+ release at 4 degrees C as well as 22 degrees C, consistent with having ion channel function. The EC50 for Ca2+ release from Xenopus oocytes is 40 microM, similar to sphingosyl-phosphocholine-mediated Ca2+ release from permeabilized mammalian cells. Because Ca2+ release is not blocked by ryanodine or La3+, the activity described here is distinct from the Ca2+ release activity of the ryanodine receptor and the inositol 1,4,5-trisphosphate receptor. The properties of SCaMPER are identical to those of the sphingolipid-gated Ca2+ channel that we have previously described. These findings suggest that SCaMPER is a sphingolipid-gated Ca2+-permeable channel and support its role as a mediator of this pathway for intracellular Ca2+ signal transduction.     

Molecular and biochemical characterization of dNOS: a Drosophila Ca2+/calmodulin-dependent nitric oxide synthase.

Nitric oxide (NO) is an intercellular messenger involved with various aspects of mammalian physiology ranging from vasodilation and macrophage cytotoxicity to neuronal transmission. NO is synthesized from L-arginine by NO synthase (NOS). Here, we report the cloning of a Drosophila NOS gene, dNOS, located at cytological position 32B. The dNOS cDNA encodes a protein of 152 kDa, with 43% amino acid sequence identity to rat neuronal NOS. Like mammalian NOSs, DNOS protein contains putative binding sites for calmodulin, FMN, FAD, and NADPH. DNOS activity is Ca2+/calmodulin dependent when expressed in cell culture. An alternative RNA splicing pattern also exists for dNOS, which is identical to that for vertebrate neuronal NOS. These structural and functional observations demonstrate remarkable conservation of NOS between vertebrates and invertebrates.     

Immunohistochemical localization of adenylyl cyclase in rat brain indicates a highly selective concentration at synapses.

Only three isoforms of adenylyl cyclase (EC 4.6.1.1) mRNAs (AC1, -2, and -5) are expressed at high levels in rat brain. AC1 occurs predominantly in hippocampus and cerebellum, AC5 is restricted to the basal ganglia, whereas AC2 is more widely expressed, but at much lower levels. The distribution and abundance of adenylyl cyclase protein were examined by immunohistochemistry with an antiserum that recognizes a peptide sequence shared by all known mammalian adenylyl cyclase isoforms. The immunoreactivity in striatum and hippocampus could be readily interpreted within the context of previous in situ hybridization studies. However, extending the information that could be gathered by comparisons with in situ hybridization analysis, it was apparent that staining was confined to the neuropil--corresponding to immunoreactive dendrites and axon terminals. Electron microscopy indicated a remarkably selective subcellular distribution of adenylyl cyclase protein. In the CA1 area of the hippocampus, the densest immunoreactivity was seen in postsynaptic densities in dendritic spine heads. Labeled presynaptic axon terminals were also observed, indicating the participation of adenylyl cyclase in the regulation of neurotransmitter release. The selective concentration of adenylyl cyclases at synaptic sites provides morphological data for understanding the pre- and postsynaptic roles of adenylyl cyclase in discrete neuronal circuits in rat brain. The apparent clustering of adenylyl cyclases, coupled with other data that suggest higher-order associations of regulatory elements including G proteins, N-methyl-D-aspartate receptors, and cAMP-dependent protein kinases, suggests not only that the primary structural information has been encoded to render the cAMP system responsive to the Ca(2+)-signaling system but also that higher-order strictures are in place to ensure that Ca2+ signals are economically delivered and propagated.     

Cloning and characterization of a sixth adenylyl cyclase isoform: types V and VI constitute a subgroup within the mammalian adenylyl cyclase family.

A sixth member of the mammalian adenylyl cyclase family has been isolated from a canine cardiac cDNA library. This isoform is more highly homologous to type V than to the other adenylyl cyclase types; sequence similarity is apparent even in the transmembrane regions where the greatest divergence among the types exists. Type VI mRNA expression is most abundant in heart and brain; however, unlike type V, a low level of expression is also observed in a variety of other tissues examined. Type VI adenylyl cyclase can be stimulated by NaF, guanosine 5'-[gamma-thio]triphosphate, and forskolin but not by Ca2+/calmodulin, whereas it is inhibited by adenosine and its analogues. Comparison of both their structural and biochemical properties suggests that types V and VI constitute a distinct subgroup of the mammalian adenylyl cyclase family.     

Molecular cloning and functional characterization of the Xenopus Ca(2+)-binding protein frequenin.

Frequenin was originally identified in Drosophila melanogaster as a Ca(2+)-binding protein facilitating transmitter release at the neuromuscular junction. We have cloned the Xenopus frequenin (Xfreq) by PCR using degenerate primers combined with low-stringency hybridization. The deduced protein has 70% identity with Drosophila frequenin and about 38-58% identity with other Ca(2+)-binding proteins. The most prominent features are the four EF-hands, Ca(2+)-binding motifs. Xfreq mRNA is abundant in the brain and virtually nondetectable from adult muscle. Western blot analysis indicated that Xfreq is highly concentrated in the adult brain and is absent from nonneural tissues such as heart and kidney. During development, the expression of the protein correlated well with the maturation of neuromuscular synapses. To determine the function of Xfreq at the developing neuromuscular junction, the recombinant protein was introduced into Xenopus embryonic spinal neurons by early blastomere injection. Synapses made by spinal neurons containing exogenous Xfreq exhibited a much higher synaptic efficacy. These results provide direct evidence that frequenin enhances transmitter release at the vertebrate neuromuscular synapse and suggest its potential role in synaptic development and plasticity.     

Age-related alterations in adenylyl cyclase system of rat hearts.

Cardiac membranes from 26-, 52- and 104-week-old Wistar rats were used to investigate the age-related alterations in the beta-adrenergic receptor-adenylyl cyclase system. The densities and affinities of beta-adrenoceptors did not change with aging. There were no significant changes in the total amount of stimulatory G-protein (Gs), and in Gs activity measured in a reconstitution assay using human platelet membranes. The major isoform of Gs alpha, however, changed from a 45,000 to 52,000 dalton peptide with aging. The total amount of pertussis toxin substrates (Gi2 and Go) decreased significantly with aging. This finding was supported by the fact that pertussis toxin-induced potentiation of adenylyl cyclase activity was markedly reduced in the aged group. The activity of catalytic protein assessed by forskolin-stimulated adenylyl cyclase activity was decreased at 104 weeks. On the other hand, GTP analogue-stimulated adenylyl cyclase activity was significantly potentiated in the same group. These results suggest that the decreased sensitivity to catecholamines observed in aged hearts is mainly due to a dysfunction of catalytic protein, and that decreased Gi activity partially compensates for this catalytic dysfunction.     

Enhanced cardiac function in transgenic mice expressing a Ca(2+)-stimulated adenylyl cyclase

The predominant functional adenylyl cyclases normally expressed in cardiac tissue and coupled to beta-adrenergic receptors are inhibited by micromolar Ca(2+) concentration. To modify the overall balance of activities, we have generated transgenic mice expressing the Ca(2+)-stimulatable adenylyl cyclase type 8 (AC8) specifically in the heart. AC activity is increased by at least 7-fold in heart membranes from transgenic animals and is stimulated by Ca(2+) in the same range of concentration that inhibits the endogenous activity. Moreover, the in vivo basal protein kinase A activity was augmented 4-fold. Overexpression of AC8 in the heart has no detrimental consequences on global cardiac function. Basal heart rate and contractile function, measured by noninvasive echocardiography, were unchanged. In contrast, on release of parasympathetic tone, the intrinsic contractility is heightened and unresponsive to further beta-adrenergic receptor stimulation. AC8 transgenic mice thus represent an original model to investigate the relative influence of Ca(2+) and cAMP on cardiac function within a phenotype of enhanced cardiac contractility and relaxation.     

The adenylyl cyclase gene from Schizosaccharomyces pombe.

We cloned the adenylyl cyclase gene from the fission yeast Schizosaccharomyces pombe using low-stringency hybridization to the Saccharomyces cerevisiae adenylyl cyclase gene. The Sc. pombe gene encodes a 1692-amino acid-residue protein. The identity of this gene was confirmed by studies of its expression in Sa. cerevisiae. Expression of the carboxyl-terminal region of the Sc. pombe adenylyl cyclase protein will suppress a temperature-sensitive mutation in the Sa. cerevisiae adenylyl cyclase gene. Furthermore, Sa. cerevisiae that lack their endogenous adenylyl cyclase gene and express the carboxyl-terminal region of the Sc. pombe adenylyl cyclase protein have measurable adenylyl cyclase activity. The carboxyl-terminal region of this protein has strong homology with the catalytic domain of the Sa. cerevisiae adenylyl cyclase. Also, Sc. pombe adenylyl cyclase, like Sa. cerevisiae adenylyl cyclase, contains a tandemly repeated motif rich in leucine. Neither yeast protein is particularly homologous to the recently cloned Gs-responsive mammalian adenylyl cyclase [Krupinski, J., Coussen, F., Bakalyar, H. A., Tang, W.-J., Feinstein, P. G., Orth, K., Slaughter, C., Reed, R. R. & Gilman, A. G. (1989) Science 244, 1558-1564].     

Ca2+-activated adenylyl cyclase 1 introduces Ca2+-dependence to beta-adrenergic stimulation of HCN2 current

Previous observations show that β-adrenergic modulation of pacemaker current (I(f)) in sinoatrial node (SAN) cells is impaired by disruption of normal Ca(2+)-homeostasis with ryanodine or BAPTA. Recently, the presence of Ca(2+)-activated adenylyl cyclase (AC) 1 was reported in SAN, and was proposed as a possible mechanism of Ca(2+)-dependence of β-adrenergic modulation. However, direct evidence that pacemaker (HCN) channels can be regulated by Ca(2+)-activated AC and that such regulation introduces Ca(2+) dependence, is lacking. Here we co-expressed AC1 or AC6 with HCN2 in neonatal rat ventricular myocytes, which lack AC1. Although both isoforms have equivalent expression level and ability to interact with HCN2, only AC1 increases intracellular cAMP content, accelerates spontaneous beating rate and modifies HCN2 biophysics. Measured HCN2 current in the AC1 group activated ~10mV more positive than in GFP or AC6. The β-adrenergic agonist isoproterenol induced a further positive shift under control conditions, but failed to do so after pretreatment with the Ca(2+) chelator BAPTA. In the AC6 group, isoproterenol shifted the HCN2 activation relation to a similar extent in the absence and presence of BAPTA. Thus, AC1 but not AC6 over-expression introduces Ca(2+)-sensitivity to the β-adrenergic response of HCN2. These results demonstrate physical and functional interaction between AC isoforms and the HCN2 pacemaker channel and support a key role of Ca(2+) activated AC1 as a molecular mechanism in Ca(2+)-dependent modulation of β-adrenergic response of heart rate.     

Phorbol ester-induced stimulation and phosphorylation of adenylyl cyclase 2.

Adenylyl cyclase 2 was expressed in Sf9 cells by recombinant baculovirus infection. Phorbol 12-myristate 13-acetate (PMA) treatment of cells expressing adenylyl cyclase 2 (AC2) increased basal activity. This increase was blocked by staurosporine, a protein kinase C inhibitor. PMA treatment increased Vmax without affecting Km. Greatest increase in basal activity was seen at physiologically relevant Mg2+ concentrations. PMA treatment did not alter sensitivity to guanine nucleotide stimulatory factor (Gs) but enhanced stimulation at all concentrations of activated Gs alpha subunit tested. AC2 was tagged at the N terminus with an 8-amino acid epitope. Epitope-tagged AC2 was purified to apparent homogeneity in a single step by using an antiepitope antibody-affinity column. The eluate was resolved by SDS/PAGE. Silver staining of the gel showed a 106-kDa band. The purified protein was recognized by antipeptide antibody against a region common to all mammalian adenylyl cyclases. The epitope-tagged enzyme expressed in Sf9 cells was also stimulated by PMA. When cells were labeled with 32P and treated with PMA, a 3-fold increase in 32P incorporation of purified epitope-tagged AC2 was observed. We conclude that activation of protein kinase C results in phosphorylation and stimulation of AC2, a cell-surface G protein effector enzyme. Thus, covalent modification of cell-surface effectors may provide an independent mode for signal transmission through G protein pathways.     

Molecular cloning and amplification of the adenylate cyclase gene.

A segment of DNA containing cya, the gene for adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1], has been isolated from Salmonella typhimurium. The phage lambda gt4 was used as a cloning vector and adenylate cyclase-positive hybrid phages were isolated that complemented adenylate cyclase-negative bacteria. The cloned DNA fragment encodes a polypeptide of molecular weight 81,000 that gives rise to adenylate cyclase activity. This protein represents a functional mutant of the bacterial adenylate cyclase. When the cya gene was amplified by inserting into a multicopy plasmid, the enzyme activity was overproduced 20-fold, but the cyclic AMP level increased only 60%, suggesting several probable regulatory mechanisms. Overproduction of enzymes by recombinant DNA techniques can be a useful probe of relationships in the metabolizing organism in vivo.     

Activation of rat gastric mucosal adenylyl cyclase by secretory inhibitors.

Prostaglandin (PG) E1, E2, A1, and A2 stimulated rat gastric corpus mucosal membrane adenylyl cyclase activity. PGE1 (Kalpha congruent to 8 muM) affected the maximum velocity but not the affinity of the enzyme for ATP and maximum PGE1 activation was not affected by histamine H1 or H2 receptor antagonists. 5'-Guanylyl-diphosphoimide (Gpp(NH)p), but not GTP, stimulated both the basal and PGE1-stimulated adenylyl cyclase activities, although the percentage stimulation by maximal PGE was the same with or without Gpp(NH)p. NaF stimulation was also additive to that of PGE1. Secretin also stimulated gastric mucosal adenylyl cyclase activity (Kalpha congruent to 30 nM). Maximal secretin activation was not additive to that of PGE1, suggesting a coupling to the same adenylyl cyclase catalytic site. These studies suggest that mucosal membranes may contain beta-adrenergic receptors. The adenylyl cyclase activating agents used in this study, PGE1, secretin, and the catecholamines, are all known inhibitors of gastric acid secretion, suggesting a possible involvement of cyclic AMP in the inhibition of acid secretion in the rat stomach.     

Norepinephrine inhibits glucose-stimulated, Ca2+-independent insulin release independently from its action on adenylyl cyclase

Inhibition of insulin release by norepinephrine has been attributed to activation of ATP-sensitive K+ channels, inactivation of voltage-dependent Ca2+ channels, and inhibition of adenylyl cyclase. However, direct inhibitory action of norepinephrine at a distal site of stimulus-secretion coupling has also been suggested. To obtain more direct evidence for norepinephrine inhibition of insulin release at a distal site, we performed experiments in intact, non-permeabilized beta cells. In rat pancreatic islets, a combination of glucose, phorbol ester and forskolin under stringent Ca2+-free conditions was used as a trigger of insulin exocytosis at a distal site. Norepinephrine inhibited this Ca2+-independent insulin release in a concentration-dependent manner, with an IC50 of 50 nM. The inhibition was complete, reversible, and pertussis toxin-sensitive, and not associated with any reduction of cAMP content in the islet cells. In conclusion, norepinephrine strongly, yet reversibly, inhibits insulin release in intact beta cells at a late step of exocytosis, through pertussis toxin-sensitive, G protein-mediated mechanism(s).