Masaru Kawamura

Expression of functional (Na+ + K+)-ATPase from cloned cDNAs

Functional (Na+ + K+)‐ATPase is formed in Xenopus oocytes injected with α‐ and β‐subunit‐specific mRNAs derived from cloned Torpedo californica cDNAs. Both the mRNAs are required for the expression of functional (Na+ + K+)‐ATPase.

Site-directed mutagenesis of Asp-376, the catalytic phosphorylation site, and Lys-507, the putative ATP-binding site, of the α-subunit of Torpedo californicaNa+K+-ATPase

Point mutations of Asp-376 of the alpha-subunit of Torpedo californica Na+/K(+)-ATPase (the site of phosphorylation during the catalytic cycle) to Asn, Glu or Thr led to virtual abolishment of Na+/K(+)-ATPase activity and ouabain-binding capacity. Replacement of Lys-507 of the same subunit (the putative ATP-binding site) by Met resulted in decreases in Na+/K(+)-ATPase activity and ouabain-binding capacity. These results are in agreement with those reported for rabbit sarcoplasmic reticulum Ca2(+)-ATPase (Maruyama, K. and MacLennan, D.H. (1988) Proc. Natl. Acad. Sci. USA 85, 3314-3318).

Adrenomedullin-sensitive receptors are preferentially expressed in cultured rat mesangial cells

By using cultured rat mesangial cells, we compared the effects on cyclic nucleotide levels of adrenomedullin with those of the structurally related peptides, calcitonin gene-related peptide (CGRP) and amylin. Adrenomedullin potently increased cAMP levels 7-fold in a time- and concentration-dependent manner. Its EC50 was 3 x 10(-9) M. CGRP was less potent (2-fold) with an EC50 of 10(-7) M, and amylin had no effect on cAMP levels. All three peptides failed to increase cGMP levels. Treatment of cells with near maximal concentrations of adrenomedullin (10(-7) M) and CGRP (10(-6) M) had no additive effect on cAMP levels. Human adrenomedullin-(22-52)-NH2, a putative adrenomedullin receptor antagonist, inhibited the production of cAMP elicited by adrenomedullin (IC50: 7 x 10(-8) M) and CGRP (IC50: 5 x 10(-8) M). Human CGRP-(8-37), a CGRP receptor antagonist, conversely, reduced the cAMP elevation caused by these peptides with a lower potency (IC50: 10(-6) M for both peptides). This demonstrated that human adrenomedullin-(22-52)-NH2 was a more effective antagonist for adrenomedullin- and CGRP-specific receptors than human CGRP-(8-37). Results suggest that receptors sensitive to adrenomedullin are preferentially expressed in cultured rat mesangial cells. Immunohistochemical study showed almost no immunoreactive adrenomedullin and CGRP, if any, in the cells. Adrenomedullin may regulate mesangial function as either a paracrine or circulating hormone via a cAMP- but not a cGMP-dependent mechanism.

KETAMINE INTERACTS WITH THE NORADRENALINE TRANSPORTER AT A SITE PARTLY OVERLAPPING THE DESIPRAMINE BINDING SITE

Effects of the intravenous anaesthetic ketamine on the desipramine-sensitive noradrenaline transporter (NAT) were examined in cultured bovine adrenal medullary cells and in transfected Xenopus laevis oocytes expressing the bovine NAT (bNAT). Incubation (1–3 h) of adrenal medullary cells with ketamine (10–300 µM) caused an increase in appearance of catecholamines in culture medium. Ketamine (10–1000 µM) inhibited desipramine-sensitive uptake of [3H] noradrenaline (NA) (IC50=97 µM). Saturation analysis showed that ketamine reduced Vmax of [3H]NA uptake without changing Km, indicating a non-competitive inhibition. Other inhibitors of NAT, namely cocaine and desipramine, showed a competitive inhibition of [3H]NA uptake while a derivative of ketamine, phencyclidine, showed a mixed type of inhibition. Ketamine (10–1000 µM) also inhibited the specific binding of [3H]desipramine to plasma membranes isolated from bovine adrenal medulla. Scatchard analysis of [3H]desipramine binding revealed that ketamine increased Kd without altering Bmax, indicating a competitive inhibition. In transfected Xenopus oocytes expressing the bNAT, ketamine attenuated [3H]NA uptake with a kinetic characteristic similar to that of cultured adrenal medullary cells. These findings are compatible with the idea that ketamine non-competitively inhibits the transport of NA by interacting with a site which partly overlaps the desipramine binding site on the NAT.

Voltage-dependent inhibition of the sodium pump by external sodium: Species differences and possible role of the N-terminus of the α-subunit

Currents generated by the Na+/K+ ATPase were measured under voltage clamp in oocytes of Xenopus laevis. The dependence of pump current on external [Na+] was investigated for the endogenous Xenopus pump as well as for wild-type and mutated pumps of electroplax of Torpedo californica expressed in the oocytes. The mutants had α-subunits truncated before position Lys28 (αΔK28) or Thr29 (αΔT29) of the N-terminus. The currents generated by all variants of pump molecules in the presence of 5 mM K+ show voltage-dependent inhibition by external [Na+]. The apparent K1 values increase with membrane depolarisation, and the potential dependence can be described by the movement of effective charges in the electrical potential gradient across the membrane. Taking into account Na+-K+ competition for external binding to the E2P form, apparent K1 values and effective charges for the interaction of the Na+ ions with the E2P form can be estimated. For the Xenopus pump the effective charge amounts to 1.1 of an elementary charge and the K1 value at 0 mV to 44 mM. For the wild-type Torpedo pump, the analysis yields values of 0.73 of an elementary charge and 133 mM, respectively. Truncation at the N-terminus removing a lysinerich cluster of the a-subunit of the Torpedo pump leads to an increase of the effective charge and decrease of the K1 value. For αΔK28, values of 0.83 of an elementary charge and 117 mM are obtained, respectively. If LyS28 is included in the truncation (α·T29), the effective charge increases to 1.5 of an elementary charge and the apparent K1 value is reduced to 107 mM. The K, values for pump inhibition by external Na+, calculated by taking into account Na+-K+ competition, are smaller than the K/12 values determined in the presence of 5 mM [K+]. The difference is more pronounced for those pump variants that have higher Km, values. The variations of the parameters describing inhibition by external [Na+] are qualitatively similar to those described for the stimulation of the pumps by external [K+] in the absence of extracellular [Na+]. The observations may be explained by an acess channel within the membrane dielectric that has to be passed by the external Na+ and K+ ions to reach or leave their binding sites. The potential-dependent access and/or the interaction with the binding sites shows species differences and is affected by cytoplasmic lysine residues in the N-terminus.

Inhibitory effects of clozapine and other antipsychotic drugs on noradrenaline transporter in cultured bovine adrenal medullary cells

Abstract The effects of clozapine and other antipsychotic drugs on noradrenaline (NA) transport were examined in cultured bovine adrenal medullary cells and in transfected Xenopus laevis oocytes expressing the bovine NA transporter. Incubation of adrenal medullary cells with clozapine (30–1000 ng/ml) inhibited desipramine (DMI)-sensitive uptake of [3H]NA in a concentration-dependent manner (IC50=110 ng/ml or 336 nM). Other antipsychotic drugs such as haloperidol, chlorpromazine, and risperidone also decreased [3H]NA uptake (IC50= 144, 220, and 210 ng/ml or 383, 690, and 512 nM, respectively). Eadie-Hofstee analysis showed that clozapine reduced Vmax of uptake of [3H]NA and increased Km. Furthermore, clozapine inhibited specific binding of [3H]DMI to plasma membranes isolated from bovine adrenal medulla (IC50=48 ng/ml or 146 nM). Scatchard plot analysis of [3H]DMI binding revealed that clozapine decreased both Bmax and Kd. Other antipsychotic drugs, including haloperidol, chlorpromazine, and risperidone, also reduced [3H]DMI binding to the membranes. In transfected Xenopus oocytes expressing the bovine NA transporter, clozapine inhibited [3H]NA uptake in a concentration-dependent manner similar to that observed in adrenal medullary cells. These results suggest that clozapine and haloperidol directly inhibit transport of NA by acting on the site of an NA transporter that influences both substrate transport and binding of tricyclic antidepressants.

Adrenomedullin increases cyclic AMP more potently than CGRP and amylin in rat renal tubular basolateral membranes

In rat renal tubular basolateral membranes, the potency to increase cAMP of adrenomedullin (AM), a novel vasorelaxant peptide originally isolated from human pheochromocytoma, was compared with those of calcitonin gene-related peptide (CGRP) and amylin. Although all three peptides raised cAMP in a time- and concentration-dependent manner with a 4-fold increase at 10(-6)-10(-5) M, the EC50 value (10(-9) M) of AM was 100-fold smaller than those of CGRP and amylin. CGRP[8-37], an antagonist for CGRP receptors, attenuated cAMP elevation induced by these peptides with the essentially similar concentration-inhibition curves. These results suggest that the receptors for AM, CGRP and amylin share a common structural homology, and that the receptors sensitive to AM are preferentially expressed in renal tubular basolateral membranes.

Assembly of the α-subunit of Torpedo californica Na+/K+-ATPase with its pre-existing β-subunit in Xenopus oocytes

Abstract The α- and β-subunits of Torpedo californica Na+/K+-ATPase were expressed in turn in single oocytes by alternately microinjection the specific mRNAs for the α- and β-subunits. The mRNA first injected was degraded prior to the injection of the second mRNA by injecting the antisense oligonucleotide specific for the first mRNA. The pre-existing β-subunit, which had been synthesized by injecting mRNA for the β-subunit, could assemble with the α-subunit expressed later in the single oocytes and the resulting αβ complex acquired both ouabain-binding and Na+/K+-ATPase activities. On the other hand, formation of the αβ complex was not detected when the α-subunit was expressed first, followed by the β-subunit. These data suggest that the β-subunit acts as a receptor or a stabilizer for the α-subunit upon the biogenesis of Na+/K+-ATPase.

Functional activity of oligosaccharide-deficient (Na,K)ATPase expressed in Xenopus oocytes

(Na,K)ATPase from Torpedo californica was expressed in Xenopus laevis oocytes in the presence of tunicamycin by injecting mRNAs for the α‐ and β‐subunits derived from the cloned cDNAs into the oocytes. The oligosaccharide‐deficient ATPase thus synthesized was transported to the oocytes plasma membrane, where it exhibited virtually the same ATPase activity, ouabain‐binding capacity and 86Rb+ transport activity as the fully glycosylated enzyme. We conclude that the oligosaccharide chains on the β‐subunit has no effect on the catalytic activities of (Na,K)ATPase.

The functional roles of disulfide bonds in the β-subunit of (Na,K)ATPase as studied by site-directed mutagenesis

The β‐subunit of Torpedo californica (Na,K)ATPase contains seven cysteine residues; one (Cys46) is in the single transmembrane segment and the other six (Cys127, Cys150, Cys160, Cys176, Cys215 and Cys278) are in the extracellular domain and form three highly conserved disulfide bonds. Aβ‐subunit mutant with replacement of Cys46 by Ser could assemble with the α‐subunit, and the resulting αβ‐complex was catalytically active. Mutants in which either the N‐terminal side or both Cys residues of the Cys127‐Cys150 bond were replaced by Ser could also tightly assemble with the α‐subunit, but the resulting αβ‐complex was catalytically inactive. On the other hand, disruption of either the Cys160‐Cys176 or Cys215‐Cys278 bond by substituting the N‐terminal side only or both Cys residues with Ser led to a β‐subunit that could not assemble with the α‐subunit. We conclude that the structure of the β‐subunit around the Cys160‐Cys176 and Cys215‐Cys278 loops is indispensable for assembly with the α‐subunit, whereas the Cys127‐Cys150 loop is not essential for assembly but is required for enzyme activity.