Protein kinase G II-mediated proliferative effects in human cultured prostatic stromal cells

This study investigates the effect of protein kinase G (PKG) activation upon proliferation of human cultured prostatic stromal cells. The PKG II activator (8-pCPT-cGMP; IC50 of 113+/-42 nM) and the phosphodiesterase inhibitor, zaprinast (up to 50 microM), but not the PKG I isoform activators (APT-cGMP and PET-cGMP), reduced foetal calf serum-stimulated proliferation. The effect of 8-pCPT-cGMP (30 microM) was blocked by Rp-8-Br-cGMPS (5 microM) and Rp-8-pCPT-cGMP (5 microM), but not Rp-cAMPS (5 microM). 8-pCPT-cGMP (30 microM) and zaprinast (50 microM), but not PET-cGMP (30 microM), caused a significant increase in atypical nuclei and an increase in annexin-V staining. These data indicate that activation of PKG II induces apoptosis of human cultured prostatic stromal cells.     

Role of K(ATP) channels in protection against neuronal excitatory insults

ATP-sensitive K⁺ (K_ATP) channels that are gated by intracellular ATP/ADP concentrations are a unique subtype of potassium channels and play an essential role in coupling intracellular metabolic events to electrical activity. Opening of K_ATP channels during energy deficits in the CNS induces efflux of potassium ions and in turn hyperpolarizes neurons. Thus, activation of K_ATP channels is thought to be able to counteract excitatory insults and protect against neuronal death. In this review, we bring together recent studies about what kinds of molecules are needed to build and regulate arrays of K_ATP channel functions in the CNS neurons. We propose a model to explain how K_ATP channel activation regulates glutamate release from the pre-synaptic terminals and how this regulation protects against ischemic neuronal injury and epilepsy.     

Protein kinase C-mediated tyrosine phosphorylation of paxillin and focal adhesion kinase requires cytoskeletal integrity and is uncoupled to mitogen-activated protein kinase activation in human hepatoma cells

Treatment of cultured human hepatoma HepG2 cells with the protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), results in an increase in tyrosine phosphorylation of several proteins, including the focal adhesion kinase (FAK) and paxillin using anti-phosphotyrosine Western blotting and immunoprecipitation. However, when cells are in suspension or in the presence of cytochalasin D which disrupts the intracellular network of actin microfilaments, TPA loses its ability to stimulate tyrosine phosphorylation of FAK and paxillin but it still activates mitogen-activated protein kinase (MAPK) and induces PKC translocation from cytosol to the membrane in HepG2 cells. On the other hand, PD98059, a specific inhibitor of mitogen-activated protein kinase kinase, blocks TPA-induced MAPK activation but has no effect on TPA-induced tyrosine phosphorylation. Our findings suggest that TPA-induced tyrosine phosphorylation of FAK and paxillin in human hepatoma cells is PKC dependent and requires the integrity of the cell cytoskeleton but is uncoupled to the signal transduction pathway of PKC leading to the translocation of PKC and MAPK activation.     

Identification and activation of mitogen-activated protein (MAP) kinase in normal human osteoblastic and bone marrow stromal cells: Attenuation of MAP kinase activation by cAMP,parathyroid hormone and forskolin

The mitogen-activated protein (MAP) kinases (p44^mapk and p42^mapk), also known as extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), are activated in response to a variety of extracellular signals, including growth factors, hormones and, neurotransmitters. We have investigated MAP kinase signal transduction pathways in normal human osteoblastic cells. Normal human bone marrow stromal (HBMS), osteoblastic (HOB), and human (TE85, MG-63, SaOS-2), rat (ROS 17/2.8, UMR-106) and mouse (MC3T3-E1) osteoblastic cell lines contained immunodetectable p44^mapk/ERK1 and p42^mapk/ERK2. MAP kinase activity was measured by 'in-gel' assay myelin basic protein as the substrate. Mainly ERK2 was rapidly activated (within 10 min) by bFGF, IGF-I and PDGF-BB in normal HOB, HBMS and human osteosarcoma cells, whereas both ERK1 and ERK2 were activated by growth factors in rat osteoblast-like cell lines, ROS 17/2.8 and UMR-106. The ERK1 activation was greater than the ERK2 in ROS 17/2.8 cells. Furthermore, ERK2 was also activated by bFGF and PDGF-BB in the mouse osteoblastic cell line, MC3T3-E1. This is the first demonstration of inter-species differences in the activation of MAP kinases in osteoblastic cells. Cyclic AMP derivatives or cAMP generating agents such as PTH and forskolin inhibited ERK2 activation by bFGF and PDGF-BB suggesting a 'cross-talk' between the two different signalling pathways activated by receptor tyrosine kinases and cAMP-dependent protein kinase. The accumulated results also suggest that the MAP kinases may be involved in mediating mitogenic and other biological actions of bFGF, IGF-I and PDGF-BB in normal human osteoblastic and bone marrow stromal cells.     

Pathophysiology of voltage-gated K channels in vascular smooth muscle cells: Modulation by protein kinases

In this review, the pathological alteration and clinical relevance of voltage-gated K⁺ (Kv) channels and their specific regulation by protein kinase-dependent signaling in vascular smooth muscle cells are described, particularly focusing on the pulmonary vasculature. The physiological relevance, channel characteristics, pharmacological modulation, and expression of Kv channels vary between different arterial beds and between subdivisions of arteries within those vascular beds. Although detailed signaling cascades regulating Kv channels are not clearly elucidated, it is known that the Kv channels in vascular smooth muscle cells can be tightly regulated by protein kinases C (PKC) and A (PKA). Alterations in Kv channel expression and function has been noted in pathological and pathophysiological conditions including hypertension (pulmonary and systemic), in diabetes and in individuals subjected to prolonged hypoxia (high altitude living). Vascular Kv channels are potential therapeutic targets in diseases such as pulmonary arterial hypertension and, therefore, it is important to understand the specific pharmacological modulation of Kv channel isoforms in different vascular beds.     

Protein kinase A-dependent activation of inward rectifier potassium channels by adenosine in rabbit coronary smooth muscle cells

We studied the effect of adenosine on the Ba(2+)-sensitive K(IR) channels in the smooth muscle cells isolated from the small-diameter (<100microm) coronary arteries of rabbit. Adenosine increased K(IR) currents in concentration-dependent manner (EC(50)=9.4+/-1.4microM, maximum increase of 153%). The adenosine-induced stimulation of K(IR) current was blocked by adenylyl cyclase inhibitor, SQ22536 and was mimicked by adenylyl cyclase activator, forskolin. The adenosine-induced increase of current was blocked by cyclic AMP-dependent protein kinase (PKA) inhibitors, KT 5720 and Rp-8-CPT-cAMPs. The adenosine-induced increase of K(IR) currents was blocked by an A(3)-selective antagonist MRS1334, while the antagonists of other subtypes (DPCPX for A(1), ZM241385 for A(2A), and alloxazine for A(2B)) were all ineffective. Furthermore, an A(3)-selective agonist, 2-Cl-IB-MECA induced increase of K(IR) currents. We also examined the effect of adenosine on coronary blood flow (CBF) rate by using the Langendorff-perfused heart. In the presence of glibenclamide to exclude the effects of ATP-sensitive K(+) (K(ATP)) channels, CBF was increased by adenosine (10microM), which was blocked by the addition of Ba(2+) (50microM). Above results suggest that adenosine increases K(IR) current via A(3) subtype through the activation of PKA in rabbit small-diameter coronary arterial smooth muscle cells.     

Tyrosine kinase modulation of protein kinase C activity regulates G protein-linked Ca2+ signaling in leukemic hematopoietic cells

We have used a recombinant mouse pre-B cell line (TonB210.1, expressing Bcr/Abl under the control of an inducible promoter) and several human leukemia cell lines to study the effect of high tyrosine kinase activity on G protein-coupled receptor (GPCR) agonist-stimulated cellular Ca²⁺ release and store-operated Ca²⁺ entry (SOCE). After induction of Bcr/Abl expression, GPCR-linked SOCE increased. The effect was reverted in the presence of the specific Abl inhibitor imatinib (1 μM) and the Src inhibitor PP2 (10 μM). In leukemic cell lines constitutively expressing high tyrosine kinase activity, Ca²⁺ transients were reduced by imatinib and/or PP2. Ca²⁺ transients were enhanced by specific inhibitors of PKC subtypes and this effect was amplified by tyrosine kinase inhibition in Bcr/Abl expressing TonB210.1 and K562 cells. Under all conditions Ca²⁺ transients were essentially blocked by the PKC activator PMA. In Bcr/Abl expressing (but not in native) TonB210.1 cells, tyrosine kinase inhibitors enhanced PKCα catalytic activity and PKCα co-immunoprecipitated with Bcr/Abl.Unlike native TonB210.1 cells, Bcr/Abl expressing cells showed a high rate of cell death if Ca²⁺ influx was reduced by complexing extracellular Ca²⁺ with BAPTA. Our data suggest that tonic inhibition of PKC represents a mechanism by which high tyrosine kinase activity can enhance cellular Ca²⁺ transients and thus exert profound effects on the proliferation, apoptosis and chemotaxis of leukemic cells.     

Heterologous facilitation of G protein-activated K(+) channels by beta-adrenergic stimulation via cAMP-dependent protein kinase

To investigate possible effects of adrenergic stimulation on G protein-activated inwardly rectifying K(+) channels (GIRK), acetylcholine (ACh)-evoked K(+) current, I(KACh), was recorded from adult rat atrial cardiomyocytes using the whole cell patch clamp method and a fast perfusion system. The rise time of I(KACh ) was 0. 4 +/- 0.1 s. When isoproterenol (Iso) was applied simultaneously with ACh, an additional slow component (11.4 +/- 3.0 s) appeared, and the amplitude of the elicited I(KACh) was increased by 22.9 +/- 5.4%. Both the slow component of activation and the current increase caused by Iso were abolished by preincubation in 50 microM H89 (N-[2-((p -bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, a potent inhibitor of PKA). This heterologous facilitation of GIRK current by beta-adrenergic stimulation was further studied in Xenopus laevis oocytes coexpressing beta(2)-adrenergic receptors, m(2 )-receptors, and GIRK1/GIRK4 subunits. Both Iso and ACh elicited GIRK currents in these oocytes. Furthermore, Iso facilitated ACh currents in a way, similar to atrial cells. Cytosolic injection of 30-60 pmol cAMP, but not of Rp-cAMPS (a cAMP analogue that is inhibitory to PKA) mimicked the beta(2)-adrenergic effect. The possibility that the potentiation of GIRK currents was a result of the phosphorylation of the beta-adrenergic receptor (beta(2)AR) by PKA was excluded by using a mutant beta(2)AR in which the residues for PKA-mediated modulation were mutated. Overexpression of the alpha subunit of G proteins (Galpha(s)) led to an increase in basal as well as agonist-induced GIRK1/GIRK4 currents (inhibited by H89). At higher levels of expressed Galpha(s), GIRK currents were inhibited, presumably due to sequestration of the beta/gamma subunit dimer of G protein. GIRK1/GIRK5, GIRK1/GIRK2, and homomeric GIRK2 channels were also regulated by cAMP injections. Mutant GIRK1/GIRK4 channels in which the 40 COOH-terminal amino acids (which contain a strong PKA phosphorylation consensus site) were deleted were also modulated by cAMP injections. Hence, the structural determinant responsible is not located within this region. We conclude that, both in atrial myocytes and in Xenopus oocytes, beta-adrenergic stimulation potentiates the ACh-evoked GIRK channels via a pathway that involves PKA-catalyzed phosphorylation downstream from beta(2)AR.     

Action of imipramine on activated ATP-sensitive K(+) channels in interstitial cells of Cajal from murine small intestine

Tricyclic antidepressants have been widely used for the treatment of depression and as a therapeutic agent for the altered gastrointestinal (GI) motility of irritable bowel syndrome (IBS). The aim of this study was to clarify whether antidepressants directly modulate pacemaker currents in cultured interstitial cells of Cajal (ICC). We used the whole-cell patch-clamp techniques at 30 degrees C in cultured ICC from the mouse small intestine. Treatment of pinacidil, an ATP-sensitive K(+) channel opener, in the ICC using the current clamping mode, produced hyperpolarization of the membrane potential and decreased the amplitude of the pacemaker potentials. With the voltage clamp mode, we observed a decrease in the frequency and amplitude of pacemaker currents and increases in the resting outward currents. These effects of pinacidil on pacemaker potentials and currents were completely suppressed by glibenclamide, an ATP-sensitive K(+) channel blocker. Also, with the current clamp mode, imipramine blocked the affect of pinacidil on the pacemaker potentials. Observations of the voltage clamp mode with imipramine, desipramine and amitryptyline suppressed the action of pinacidil in the ICC. Next, we examined whether protein kinase C (PKC) and the G protein are involved in the action of imipramine on pinacidil induced pacemaker current inhibition. We used chelerythrine, a potent PKC inhibitor and GDPbetaS, a nonhydrolyzable guanosine 5-diphosphate (GDP) analogue that permanently inactivates GTP-binding proteins. We found that pretreatment with chelerythrine and intracellular application of GDPbetaS had no influence on the blocking action of imipramine on inhibited pacemaker currents by pinacidil. We conclude that imipramine inhibited the activated ATP-sensitive K(+) channels in ICC. This action does not appear to be mediated through the G protein and protein kinase C. Furthermore, this study may suggest another possible mechanism for tricyclic antidepressants related modulation of GI motility.     

κ-阿片受体激动通过激活KATP通道对大鼠腹主动脉产生舒张作用

为观察U50,488H（选择性κ－阿片受体激动剂）对大鼠腹主动态的佶张作用,并探讨其机制,实验采用离体血管灌流实验,测定血管张力的改变。结果显示：（1）U50,488H对大鼠腹主动脉具有明显的舒张作用;（2）U50,488H对大鼠腹主动脉的舒张效应部分依赖于内皮细胞的存在;（3）优降糖和格列甲嗪可明显抑制U50,488H对大鼠腹主动脉的佶张作用;（4）U50,488H的舒张血管效应与M受体、β受体、前列腺素及NO无关。结果表明,U50,488H是一种有效的扩血管物质,其舒张血管的效应具有内皮依赖性,且与KATP通道有密切关系。     

ATP-dependent activation of K(Ca) and ROMK-type K(ATP) channels in human submandibular gland ductal cells.

[Ca(2+)](i) and membrane current were measured in human submandibular gland ductal (HSG) cells to determine the regulation of salivary cell function by ATP. 1-10 microM ATP activated internal Ca(2+) release, outward Ca(2+)-dependent K(+) channel (K(Ca)), and inward store-operated Ca(2+) current (I(SOC)). The subsequent addition of 100 microM ATP activated an inwardly rectifying K(+) current, without increasing [Ca(2+)](i). The K(+) current was also stimulated by ATP in cells treated with thapsigargin in a Ca(2+)-free medium and was blocked by glibenclamide and tolbutamide, but not by charybdotoxin. This suggests the involvement of a Ca(2+)-independent, sulfonylurea-sensitive K(+) channel (K(ATP)). UTP mimicked the low [ATP] effects, while benzoyl-ATP activated internal Ca(2+) release, a Ca(2+) influx pathway, and K(Ca). Thus, ATP acts via P(2U) (P2Y(2)) and P(2Z) (P2X(7)) receptors to increase [Ca(2+)](i) and activate K(Ca), but not K(ATP). Importantly, (i) ROMK1 and the cystic fibrosis transmembrane regulator protein (but not SUR1, SUR2A, or SUR2B) and (ii) cAMP-stimulated Cl(-) and K(+) currents were detected in HSG cells. These data demonstrate for the first time that a ROMK-type K(ATP) channel is present in salivary gland duct cells that is regulated by extracellular ATP and possibly by the cystic fibrosis transmembrane regulator. This reveals a potentially novel mechanism for K(+) secretion in these cells.     

Voltage-dependent L-type Ca channels and a novel type of non-selective cation channel activated by cAMP-dependent phosphorylation in mesoderm-like (MES-1) cells

Undifferentiated P19 embryonal carcinoma cells (ECC P19), the P19-derived clonal cell lines END-2 (visceral endoderm-like), EPI-7 (epithelioid ectoderm-like), MES-1 (mesoderm-like) and a parietal yolk sac cell line (PYS-2) were used as cellular models to examine the functional expression of voltage-dependent Ca channels and other Ca-permeable cation channels at various stages of early embryonic development. Whole-cell currents were recorded by means of the patch clamp technique. Whereas more than 75% of MES-1 cells possessed Ca channel currents, neither P19, END-2, EPI-7 nor PYS-2 cells had detectable voltage-dependent inward currents. Ca channel currents of MES-1 cells were highly sensitive towards 1,4-dihydropyridines and blocked by cadmium. Adrenaline (10 μM) caused Ca channel stimulation in only 14% of MES-1 cells examined. However, in 62% of the cells adrenaline activated a linear current component which under physiological conditions reversed close to 0 mV. Removal of extracellular Na⁺ suppressed the adrenaline-induced inward current, while reducing extracellular Cl⁻ had no significant effect. These findings suggest that the adrenaline-induced current is carried through non-selective cation channels which were found to be permeable for Na⁺, K⁺, Cs⁺ å Ca²⁺. Remarkably, the intracellular signalling pathway for activation of the non-selective cation current involved the cascade of reactions leading to cAMP-dependent phosphorylation, a regulatory pathway well known for cardiac Ca channels. A possible functional role of adrenaline-induced non-selective cation currents and Ca channels in embryonal development is discussed.     

Protein kinase C inhibitors alter neurotensin receptor binding and function in prostate cancer PC3 cells

Prostate cancer PC3 cells expressed constitutive protein kinase C (PKC) activity that under basal conditions suppressed neurotensin (NT) receptor function. The endogenous PKC activity, assessed using a cell-based PKC substrate phosphorylation assay, was diminished by PKC inhibitors and enhanced by phorbol myristic acid (PMA). Accordingly, PKC inhibitors (staurosporine, Go-6976, Go-6983, Ro-318220, BIS-1, chelerythrine, rottlerin, quercetin) enhanced NT receptor binding and NT-induced inositol phosphate (IP) formation. In contrast, PMA inhibited these functions. The cells expressed conventional PKCs (, βI) and novel PKCs (δ, ε), and the effects of PKC inhibitors on NT binding were blocked by PKC downregulation. The inhibition of NT binding by PMA was enhanced by okadaic acid and blocked by PKC inhibitors. However, when some PKC inhibitors (rottlerin, BIS-1, Ro-318220, Go-69830, quercetin) were used at higher concentrations (> 2 μM), they had a different effect characterized by a dramatic increase in NT binding and an inhibition of NT-induced IP formation. The specificity of the agents implicated novel PKCs in this response and indeed, the inhibition of NT-induced IP formation was reproduced by PKCδ or PKCε knockdown. The inhibition of IP formation appeared to be specific to NT since it was not observed in response to bombesin. Scatchard analyses indicated that the PKC-directed agents modulated NT receptor affinity, not receptor number or receptor internalization. These findings suggest that PKC participates in heterologous regulation of NT receptor function by two mechanisms: a) — conventional PKCs inhibit NT receptor binding and signaling; and b) — novel PKCs maintain the ability of NT to stimulate PLC. Since NT can activate PKC upon binding to its receptor, it is possible that NT receptor is also subject to homologous regulation by PKC.     

On the mechanism of activation of protein kinase FA (an activating factor of ATP.Mg-dependent protein phosphatase) in brain myelin

Protein kinase F_A (an activating factor of ATP·Mg-dependent protein phosphatase) has been characterized to exist in two forms in the purified brain myelin. One form of kinase F_A is spontaneously active and trypsin-labile, whereas the other form of kinase F_A is inactive and trypsin-resistant, suggesting a different membrane topography with active F_A exposed on the outer face of the myelin membrane and inactivu F_Q buried within the myelin membrane. When myelin was solubilized in 1% Triton X-100, all kinase F_A became active and trypsin-labile. Phospholipid reconstitution studies further indicated that when kinase F_A was reconstituted in acidic phospholipids, such as phosphatidylinositol and phosphatidylserine, the enzyme activity was inhibited in a dose-dependent manner, suggesting that kinase F_A interacts with acidic phospholipids which inhibit its activity. Furthermore, when myelin was incubated with exogenous phospholipase C, the inactive/trypsin-resistant F_A could be converted to the active/trypsin-labile F_A in a time- and dose-dependent manner. Taken together, it is concluded that membrane phospholipids play an important role in modulating the activity of kinase F_A in the brain myelin. It is suggested that phospholipase C may mediate the activation-sequestration of inactive/trypsin-resistant kinase F_A in the brain myelin through the phospholipase C-katalyzed degradation of acidic membrane phospholipids. The activation-sequestration of protein Kinase F_A may represent one mode of control modulating the activity of kinase F_A in the central nervous system myelin.     

Diabetes and hyperglycemia impair activation of mitochondrial K(ATP) channels

Hyperglycemia is an important predictor of cardiovascular mortality in patients with diabetes. We investigated the hypothesis that diabetes or acute hyperglycemia attenuates the reduction of myocardial infarct size produced by activation of mitochondrial ATP-regulated potassium (K(ATP)) channels. Acutely instrumented barbiturate-anesthetized dogs were subjected to a 60-min period of coronary artery occlusion and 3 h of reperfusion. Myocardial infarct size (triphenyltetrazolium chloride staining) was 25 +/- 1, 28 +/- 3, and 25 +/- 1% of the area at risk (AAR) for infarction in control, diabetic (3 wk after streptozotocin-alloxan), and hyperglycemic (15% intravenous dextrose) dogs, respectively. Diazoxide (2.5 mg/kg iv) significantly decreased infarct size (10 +/- 1% of AAR, P < 0.05) but did not produce protection in the presence of diabetes (28 +/- 5%) or moderate hyperglycemia (blood glucose 310 +/- 10 mg/dl; 23 +/- 2%). The dose of diazoxide and the degree of hyperglycemia were interactive. Profound (blood glucose 574 +/- 23 mg/dl) but not moderate hyperglycemia blocked the effects of high-dose (5.0 mg/kg) diazoxide [26 +/- 3, 15 +/- 3 (P < 0.05), and 11 +/- 2% (P < 0.05), respectively]. There were no differences in systemic hemodynamics, AAR, or coronary collateral blood flow (by radioactive microspheres) between groups. The results indicate that diabetes or hyperglycemia impairs activation of mitochondrial K(ATP) channels.     

Control of K+ channels by G proteins

Heterotrimeic G proteins are thought to couple receptors to ionic channels via cytoplasmic mediators such as cGMP in the case of retinal rods, cAMP in the case of olfactory cells, and the cAMP cascade in the case of cardiac myocytes. G protein-mediated second messenger effects on K⁺ channels are dealt with elsewhere in this series. Recently, membrane-delimited pathways have been uncovered and an hypothesis proposed in which the subunits of G proteins directly couple receptors to ionic channels, particularly K⁺ channels. While direct coupling has not been proven, the membrane-delimited nature has been established for specific G proteins and their specific K⁺ channel effectors.     

Protein kinase Cdelta participates in insulin-induced activation of PKB via PDK1

PKCdelta has been shown to be activated by insulin and to interact with insulin receptor and IRS. PKB(Akt) plays an important role in glucose transport and glycogen synthesis. In this study, we investigated the possibility that PKCdelta may be involved in insulin-induced activation of PKB. Studies were conducted on primary cultures of rat skeletal muscle. PKB was activated by insulin stimulation within 5min and reached a peak by 15-30min. Insulin also increased the physical association between PKCdelta with PKB and with PDK1. The insulin-induced PKCdelta-PKB association was PI3K dependent. PKB-PKCdelta association was accounted for by the involvement of PDK1. Overexpression of dominant negative PKCdelta abrogated insulin-induced association of PKCdelta with both PKB and PDK1. Blockade of PKCdelta also decreased insulin-induced Thr308 PKB phosphorylation and PKB translocation. Moreover, PKCdelta inhibition reduced insulin-induced GSK3 phosphorylation. The results indicate that insulin-activated PKCdelta interacts with PDK1 to regulate PKB.     

Rapid effects of estrogen on G protein-coupled receptor activation of potassium channels in the central nervous system (CNS)

Estrogen rapidly alters the excitability of hypothalamic neurons that are involved in regulating numerous homeostatic functions including reproduction, stress responses, feeding and motivated behaviors. Some of the neurons include neurosecretory neurons such as gonadotropin-releasing hormone (GnRH) and dopamine neurons, and local circuitry neurons such as proopiomelanocortin (POMC) and γ-aminobutyric acid (GABA) neurons. We have elucidated several non-genomic pathways through which the steroid alters synaptic responses in these hypothalamic neurons. We have examined the modulation by estrogen of the coupling of various receptor systems to inwardly-rectifying and small-conductance, Ca²⁺-activated K⁺ (SK) channels using intracellular sharp-electrode and whole-cell recording techniques in hypothalamic slices from ovariectomized female guinea pigs. Estrogen rapidly uncouples μ-opioid receptors from G protein-gated inwardly-rectifying K⁺ (GIRK) channels in POMC neurons and GABA_B receptors from GIRK channels in dopamine neurons as manifested by a reduction in the potency of μ-opioid and GABA_B receptor agonists to hyperpolarize their respective cells. This effect is blocked by inhibitors of protein kinase A (PKA) and protein kinase C (PKC). In addition, after 24 h following steroid administration in vivo, the GABA_B/GIRK channel uncoupling observed in GABAergic neurons of the preoptic area is associated with reduced agonist efficacy. Conversely, estrogen enhances the efficacy of ₁-adrenergic receptor agonists to inhibit apamin-sensitive SK currents in these preoptic GABAergic neurons, and does so in both a rapid and sustained fashion. Finally, we observed a direct, steroid-induced hyperpolarization of GnRH neurons. These findings indicate a richly complex yet coordinated steroid modulation of K⁺ channel activity in hypothalamic (POMC, dopamine, GABA, GnRH) neurons that are involved in regulating numerous homeostatic functions.