Kazuyoshi Nakamura

Suppression by Prolactin of the Electrically Induced Erectile Response through its Direct Effect on the Corpus Cavernosum Penis in the Dog

PURPOSEnMen become impotent when exposed to hyperprolactinemia. To clarify its mechanisms the effects of intracorporal infusion of prolactin on electrically induced penile erection were evaluated in 12 male dogs.nnnMATERIALS AND METHODSnProlactin (10 micrograms./ml.) or control saline was directly infused into the corpus cavernosum penis 5 minutes before electrical pulse stimulation of the pelvic nerve and the intracorporal pressure was monitored.nnnRESULTSnIn 8 dogs erection was markedly suppressed or completely abolished by prolactin. In the remaining 4, this effect of prolactin became manifest only when the ipsilateral internal pudenal artery was ligated. Saline infusion was without effect.nnnCONCLUSIONSnAn excess of prolactin directly inhibited the smooth muscle relaxation of corpus cavernosum penis.

Role of Calcineurin-Mediated Dephosphorylation in Modulation of an Inwardly Rectifying K + Channel in Human Proximal Tubule Cells

Activity of an inwardly rectifying K+ channel with inward conductance of about 40 pS in cultured human renal proximal tubule epithelial cells (RPTECs) is regulated at least in part by protein phosphorylation and dephosphorylation. In this study, we examined involvement of calcineurin (CaN), a Ca2+/calmodulin (CaM)–dependent phosphatase, in modulating K+ channel activity. In cell-attached mode of the patch-clamp technique, application of a CaN inhibitor, cyclosporin A (CsA, 5xa0μM) or FK520 (5xa0μM), significantly suppressed channel activity. Intracellular Ca2+ concentration ([Ca2+]i) estimated by fura-2 imaging was elevated by these inhibitors. Since inhibition of CaN attenuates some dephosphorylation with increase in [Ca2+]i, we speculated that inhibiting CaN enhances Ca2+-dependent phosphorylation, which might result in channel suppression. To verify this hypothesis, we examined effects of inhibitors of PKC and Ca2+/CaM-dependent protein kinase-II (CaMKII) on CsA-induced channel suppression. Although the PKC inhibitor GF109203X (500xa0nM) did not influence the CsA-induced channel suppression, the CaMKII inhibitor KN62 (20xa0μM) prevented channel suppression, suggesting that the channel suppression resulted from CaMKII-dependent processes. Indeed, Western blot analysis showed that CsA increased phospho-CaMKII (Thr286), an activated CaMKII in inside–out patches, application of CaM (0.6xa0μM) and CaMKII (0.15 U/ml) to the bath at 10−6 M Ca2+ significantly suppressed channel activity, which was reactivated by subsequent application of CaN (800 U/ml). These results suggest that CaN plays an important role in supporting K+ channel activity in RPTECs by preventing CaMKII-dependent phosphorylation.

Interaction between Calcineurin and Ca2+/Calmodulin Kinase-II in Modulating Cellular Functions

Roles of calcineurin (CaN), a Ca2+/calmodulin- (CaM-) dependent protein phosphatase, and Ca2+/CaM-dependent protein kinase-II (CaMKII) in modulating K+ channel activity and the intracellular Ca2+ concentration ([Ca2+]i) have been investigated in renal tubule epithelial cells. The channel current through the cell membrane was recorded with the patch-clamp technique, and [Ca2+]i was monitored using fura-2 imaging. We found that a CaN-inhibitor, cyclosporin A (CyA), lowered the K+ channel activity and elevated [Ca2+]i, suggesting that CyA closes K+ channels and opens Ca2+-release channels of the cytosolic Ca2+-store. Moreover, both of these responses were blocked by KN-62, an inhibitor of CaMKII. It is suggested that the CyA-mediated response results from the activation of CaMKII. Indeed, Western blot analysis revealed that CyA increased phospho-CaMKII, an active form of CaMKII. These findings suggest that CaN-dependent dephosphorylation inhibits CaMKII-mediated phosphorylation, and the inhibition of CaN increases phospho-CaMKII, which results in the stimulation of CaMKII-dependent cellular actions.

Effects of cytokines on potassium channels in renal tubular epithelia

Renal tubular potassium (K+) channels play important roles in the formation of cell-negative potential, K+ recycling, K+ secretion, and cell volume regulation. In addition to these physiological roles, it was reported that changes in the activity of renal tubular K+ channels were involved in exacerbation of renal cell injury during ischemia and endotoxemia. Because ischemia and endotoxemia stimulate production of cytokines in immune cells and renal tubular cells, it is possible that cytokines would affect K+ channel activity. Although the regulatory mechanisms of renal tubular K+ channels have extensively been studied, little information is available about the effects of cytokines on these K+ channels. The first report was that tumor necrosis factor acutely stimulated the single channel activity of the 70xa0pS K+ channel in the rat thick ascending limb through activation of tyrosine phosphatase. Recently, it was also reported that interferon-γ (IFN-γ) and interleukin-1β (IL-1β) modulated the activity of the 40xa0pS K+ channel in cultured human proximal tubule cells. IFN-γ exhibited a delayed suppression and an acute stimulation of K+ channel activity, whereas IL-1β acutely suppressed the channel activity. Furthermore, these cytokines suppressed gene expression of the renal outer medullary potassium channel. The renal tubular K+ channels are functionally coupled to the coexisting transporters. Therefore, the effects of cytokines on renal tubular transporter activity should also be taken into account, when interpreting their effects on K+ channel activity.

Delayed and acute effects of interferon-γ on activity of an inwardly rectifying K+ channel in cultured human proximal tubule cells

The activity of an inwardly rectifying K(+) channel in cultured human renal proximal tubule cells (RPTECs) is stimulated and inhibited by nitric oxide (NO) at low and high concentrations, respectively. In this study, we investigated the effects of IFN-gamma, one of the cytokines which affect the expression of inducible NO synthase (iNOS), on intracellular NO and channel activity of RPTECs, using RT-PCR, NO imaging, and the cell-attached mode of the patch-clamp technique. Prolonged incubation (24 h) of cells with IFN-gamma (20 ng/ml) enhanced iNOS mRNA expression and NO production. In these cells, a NOS inhibitor, N(omega)-nitro-l-arginine methyl ester (l-NAME; 100 microM), elevated channel activity, suggesting that NO production was so high as to suppress the channel. This indicated that IFN-gamma would chronically suppress channel activity by enhancing NO production. Acute effects of IFN-gamma was also examined in control cells. Simple addition of IFN-gamma (20 ng/ml) to the bath acutely stimulated channel activity, which was abolished by inhibitors of IFN-gamma receptor-associated Janus-activated kinase [P6 (1 microM) and AG490 (10 microM)]. However, l-NAME did not block the acute effect of IFN-gamma. Indeed, IFN-gamma did not acutely affect NO production. Moreover, the acute effect was not blocked by inhibition of PKA, PKG, and phosphatidylinositol 3-kinase (PI3K). We conclude that IFN-gamma exerted a delayed suppressive effect on K(+) channel activity by enhancing iNOS expression and an acute stimulatory effect, which was independent of either NO pathways or phosphorylation processes mediated by PKA, PKG, and PI3K in RPTECs.

Studies on the Site of Ethanol Action in Inducing Prolactin Release in Male Rats

Hypersecretion of prolactin (PRL) has been implicated as one of the factors that mediate ethanol-induced hypogonadism, but the site(s) in the central nervous system where ethanol acts to lead to the stimulation of PRL secretion is unknown. To clarify the site(s) of ethanol action, medial basal hypothalamic deafferentation (MBHD) or medial basal hypothalamic ablation (MBHA) were performed stereotaxically in male rats, and their PRL secretory capacity in response to acute ethanol administration was compared with that of intact or sham-operated controls. In intact control rats, plasma immunoreactive PRL concentration increased markedly (P < .001 v saline injection) following ethanol 400 to 500 mg/100 g body weight (BW) intraperitoneally (IP). The PRL response was dose-related and reached a maximum plateau level at 15 minutes. Plasma PRL returned to a near-basal level by 60 minutes. The response was blocked completely (P < .001) by pretreatment with dopamine (1 mg per rat), a specific inhibitor of adenohypophyseal PRL secretion. In sham-operated rats and in MBHD and MBHA rats, ethanol (500 mg/100 g BW IP) induced a significant (P < .001 to .05) elevation of PRL relative to the respective saline treatment. The basal level was significantly (P < .005) lower in the MBHD group (5.3 +/- 0.9 ng/mL) and significantly (P < .001) higher in the MBHA group (101.1 +/- 15.7 ng/mL) than in the sham group (17.2 +/- 5.9 ng/mL). These results suggest the following: (1) acute ethanol administration stimulates PRL secretion from the pituitary in a dose-related manner, (2) ethanol appears to have direct stimulatory effects on adenohypophyseal PRL secretion, and (3) extrahypothalamic brain areas exert a stimulatory influence and the hypothalamus an inhibitory influence on basal PRL secretion.

A nicardipine-sensitive Ca2+ entry contributes to the hypotonicity-induced increase in [Ca2+]i of principal cells in rat cortical collecting duct.

We examined the mechanisms involved in the [Ca(2+)](i) response to the extracellular hypotonicity in the principal cells of freshly isolated rat cortical collecting duct (CCD), using Fura-2/AM fluorescence imaging. Reduction of extracellular osmolality from 305 (control) to 195 mosmol/kgH(2)O (hypotonic) evoked transient increase in [Ca(2+)](i) of principal cells of rat CCDs. The [Ca(2+)](i) increase was markedly attenuated by the removal of extracellular Ca(2+)(.) The application of a P(2) purinoceptor antagonist, suramin failed to inhibit the hypotonicity-induced [Ca(2+)](i) increase. The [Ca(2+)](i) increase in response to extracellular hypotonicity was not influenced by application of Gd(3+) and ruthenium red. On the other hand, a voltage-gated Ca(2+) channel inhibitor, nicardipine, significantly reduced the peak amplitude of [Ca(2+)](i) increase in the principal cells. In order to assess Ca(2+) entry during the hypotonic stimulation, we measured the quenching of Fura-2 fluorescence intensity by Mn(2+). The hypotonic stimulation enhanced quenching of Fura-2 fluorescence by Mn(2+), indicating that a Ca(2+)-permeable pathway was activated by the hypotonicity. The hypotonicity-mediated enhancement of Mn(2+) quenching was significantly inhibited by nicardipine. These results strongly suggested that a nicardipine-sensitive Ca(2+) entry pathway would contribute to the mechanisms underlying the hypotonicity-induced [Ca(2+)](i) elevation of principal cells in rat CCD.

Intracellular Mg2+ Influences Both Open and Closed Times of a Native Ca2+-activated BK Channel in Cultured Human Renal Proximal Tubule Cells

Effects of intracellular Mg2+ on a native Ca2+-and voltage-sensitive large-conductance K+ channel in cultured human renal proximal tubule cells were examined with the patch-clamp technique in the inside-out mode. At an intracellular concentration of Ca2+ ([Ca2+]i) of 10−5–10−4 M, addition of 1–10 mM Mg2+ increased the open probability (Po) of the channel, which shifted the Po –membrane potential (Vm) relationship to the negative voltage direction without causing an appreciable change in the gating charge (Boltzmann constant). However, the Mg2+-induced increase in Po was suppressed at a relatively low [Ca2+]i (10−5.5–10−6 M). Dwell-time histograms have revealed that addition of Mg2+ mainly increased Po by extending open times at 10−5 M Ca2+ and extending both open and closed times simultaneously at 10−5.5 M Ca2+. Since our data showed that raising the [Ca2+]i from 10−5 to 10−4 M increased Po mainly by shortening the closed time, extension of the closed time at 10−5.5 M Ca2+ would result from the Mg2+-inhibited Ca2+-dependent activation. At a constant Vm, adding Mg2+ enhanced the sigmoidicity of the Po–[Ca2+]i relationship with an increase in the Hill coefficient. These results suggest that the major action of Mg2+ on this channel is to elevate Po by lengthening the open time, while extension of the closed time at a relatively low [Ca2+]i results from a lowering of the sensitivity to Ca2+ of the channel by Mg2+, which causes the increase in the Hill coefficient.

Proinflammatory Cytokines and Potassium Channels in the Kidney

Proinflammatory cytokines affect several cell functions via receptor-mediated processes. In the kidney, functions of transporters and ion channels along the nephron are also affected by some cytokines. Among these, alteration of activity of potassium ion (K+) channels induces changes in transepithelial transport of solutes and water in the kidney, since K+ channels in tubule cells are indispensable for formation of membrane potential which serves as a driving force for the transepithelial transport. Altered K+ channel activity may be involved in renal cell dysfunction during inflammation. Although little information was available regarding the effects of proinflammatory cytokines on renal K+ channels, reports have emerged during the last decade. In human proximal tubule cells, interferon-γ showed a time-dependent biphasic effect on a 40u2009pS K+ channel, that is, delayed suppression and acute stimulation, and interleukin-1β acutely suppressed the channel activity. Transforming growth factor-β1 activated KCa3.1 K+ channel in immortalized human proximal tubule cells, which would be involved in the pathogenesis of renal fibrosis. This review discusses the effects of proinflammatory cytokines on renal K+ channels and the causal relationship between the cytokine-induced changes in K+ channel activity and renal dysfunction.

Interleukin-1β suppresses activity of an inwardly rectifying K+ channel in human renal proximal tubule cells

We investigated the effect of interleukin-1β (IL-1β) on activity of an inwardly rectifying K+ channel in cultured human proximal tubule cells (RPTECs), using the patch-clamp technique and Fura-2 Ca2+ imaging. IL-1β (15xa0pg/ml) acutely reduced K+ channel activity in cell-attached patches. This effect was blocked by the IL-1 receptor antagonist (20xa0ng/ml), an inhibitor of phospholipase C, neomycin (300xa0μM), and an inhibitor of protein kinase C (PKC), GF109203X (500xa0nM). The Fura-2 Ca2+ imaging revealed that IL-1β increased intracellular Ca2+ concentration even after removal of extracellular Ca2+, which was blocked by an inhibitor of inositol 1,4,5-trisphosphate receptors, 2-aminoethoxydiphenyl borate (2-APB, 1xa0μM). Moreover, IL-1β suppressed channel activity in the presence of 2-APB without extracellular Ca2+. These results suggest that IL-1β suppresses K+ channel activity in RPTECs through binding to its specific receptor and activation of the PKC pathway even though intracellular Ca2+ does not increase.