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The cysteine-rich LIM-only protein 4 (CRP4), a LIM-domain and zinc finger containing adapter protein, has been implicated as a downstream effector of the second messenger 3′,5′-cyclic guanosine monophosphate (cGMP) pathway in multiple cell types, including vascular smooth muscle cells (VSMCs). VSMCs and nitric oxide (NO)-induced cGMP signaling through cGMP-dependent protein kinase type I (cGKI) play fundamental roles in the physiological regulation of vascular tone and arterial blood pressure (BP). However, it remains unclear whether the vasorelaxant actions attributed to the NO/cGMP axis require CRP4. This study uses mice with a targeted deletion of the CRP4 gene (CRP4 KO) to elucidate whether cGMP-elevating agents, which are well known for their vasorelaxant properties, affect vessel tone, and thus, BP through CRP4. Cinaciguat, a NO- and heme-independent activator of the NO-sensitive (soluble) guanylyl cyclase (NO-GC) and NO-releasing agents, relaxed both CRP4-proficient and -deficient aortic ring segments pre-contracted with prostaglandin F2α. However, the magnitude of relaxation was slightly, but significantly, increased in vessels lacking CRP4. Accordingly, CRP4 KO mice presented with hypotonia at baseline, as well as a greater drop in systolic BP in response to the acute administration of cinaciguat, sodium nitroprusside, and carbachol. Mechanistically, loss of CRP4 in VSMCs reduced the Ca2+-sensitivity of the contractile apparatus, possibly involving regulatory proteins, such as myosin phosphatase targeting subunit 1 (MYPT1) and the regulatory light chain of myosin (RLC). In conclusion, the present findings confirm that the adapter protein CRP4 interacts with the NO-GC/cGMP/cGKI pathway in the vasculature. CRP4 seems to be part of a negative feedback loop that eventually fine-tunes the NO-GC/cGMP axis in VSMCs to increase myofilament Ca2+ desensitization and thereby the maximal vasorelaxant effects attained by (selected) cGMP-elevating agents.

The large-conductance voltage- and calcium-activated potassium (BK) channels are ubiquitously expressed in the brain and play an important role in the regulation of neuronal excitation. Previous work has shown that the total deletion of these channels causes an impaired motor behavior, consistent with a cerebellar dysfunction. Cellular analyses showed that a decrease in spike firing rate occurred in at least two types of cerebellar neurons, namely in Purkinje neurons (PNs) and in Golgi cells. To determine the relative role of PNs, we developed a cell-selective mouse mutant, which lacked functional BK channels exclusively in PNs. The behavioral analysis of these mice revealed clear symptoms of ataxia, indicating that the BK channels of PNs are of major importance for normal motor coordination. By using combined two-photon imaging and patch-clamp recordings in these mutant mice, we observed a unique type of synaptic dysfunction in vivo, namely a severe silencing of the climbing fiber–evoked complex spike activity. By performing targeted pharmacological manipulations combined with simultaneous patch-clamp recordings in PNs, we obtained direct evidence that this silencing of climbing fiber activity is due to a malfunction of the tripartite olivo-cerebellar feedback loop, consisting of the inhibitory synaptic connection of PNs to the deep cerebellar nuclei (DCN), followed by a projection of inhibitory DCN afferents to the inferior olive, the origin of climbing fibers. Taken together, our results establish an essential role of BK channels of PNs for both cerebellar motor coordination and feedback regulation in the olivo-cerebellar loop.

Colonic epithelial K+ secretion is a two-step transport process with initial K+ uptake over the basolateral membrane followed by K+ channel-dependent exit into the lumen. In this process the large-conductance, Ca2+-activated KCa1.1 (BK) channel has been identified as the only apparent secretory K+ channel in the apical membrane of the murine distal colon. The BK channel is responsible for both resting and Ca2+-activated colonic K+ secretion and is up-regulated by aldosterone. Agonists (e.g. adrenaline) that elevate cAMP are potent activators of distal colonic K+ secretion. However, the secretory K+ channel responsible for cAMP-induced K+ secretion remains to be defined. In this study we used the Ussing chamber to identify adrenaline-induced electrogenic K+ secretion. We found that the adrenaline-induced electrogenic ion secretion is a compound effect dominated by anion secretion and a smaller electrically opposing K+ secretion. Using tissue from (i) BK wildtype (BK+/+) and knockout (BK−/−) and (ii) cystic fibrosis transmembrane regulator (CFTR) wildtype (CFTR+/+) and knockout (CFTR−/−) mice we were able to isolate the adrenaline-induced K+ secretion. We found that adrenaline-induced K+ secretion: (1) is absent in colonic epithelia from BK−/− mice, (2) is greatly up-regulated in mice on a high K+ diet and (3) is present as sustained positive current in colonic epithelia from CFTR−/− mice. We identified two known C-terminal BK α-subunit splice variants in colonic enterocytes (STREX and ZERO). Importantly, the ZERO variant known to be activated by cAMP is differentially up-regulated in enterocytes from animals on a high K+ diet. In summary, these results strongly suggest that the adrenaline-induced distal colonic K+ secretion is mediated by the BK channel and probably involves aldosterone-induced ZERO splice variant up-regulation.

The large-conductance, voltage-dependent and Ca(2+)-dependent K(+) (BK) channel links membrane depolarization and local increases in cytosolic free Ca(2+) to hyperpolarizing K(+) outward currents, thereby controlling smooth muscle contractility. Constitutive deletion of the BK channel in mice (BK(-/-)) leads to an overactive bladder associated with increased intravesical pressure and frequent micturition, which has been revealed to be a result of detrusor muscle hyperexcitability. Interestingly, time-dependent and smooth muscle-specific deletion of the BK channel (SM-BK(-/-)) caused a more severe phenotype than displayed by constitutive BK(-/-) mice, suggesting that compensatory pathways are active in the latter. In detrusor muscle of BK(-/-) but not SM-BK(-/-) mice, we found reduced L-type Ca(2+) current density and increased expression of cAMP kinase (protein kinase A; PKA), as compared with control mice. Increased expression of PKA in BK(-/-) mice was accompanied by enhanced beta-adrenoceptor/cAMP-mediated suppression of contractions by isoproterenol. This effect was attenuated by about 60-70% in SM-BK(-/-) mice. However, the Rp isomer of adenosine-3',5'-cyclic monophosphorothioate, a blocker of PKA, only partially inhibited enhanced cAMP signaling in BK(-/-) detrusor muscle, suggesting the existence of additional compensatory pathways. To this end, proteome analysis of BK(-/-) urinary bladder tissue was performed, and revealed additional compensatory regulated proteins. Thus, constitutive and inducible deletion of BK channel activity unmasks compensatory mechanisms that are relevant for urinary bladder relaxation.

Mammalian K+ homeostasis results from highly regulated renal and intestinal absorption and secretion, which balances the unregulated K+ intake. Aldosterone is known to enhance both renal and colonic K+ secretion. In mouse distal colon K+ secretion occurs exclusively via luminal KCa1.1 (BK) channels. Here we investigate if aldosterone stimulates colonic K+ secretion via BK channels. Luminal Ba2+ and iberiotoxin (IBTX)-sensitive electrogenic K+ secretion was measured in Ussing chambers. In vivo aldosterone was augmented via a high K+ diet. High K+ diet led to a 2-fold increase of luminal Ba2+ and IBTX-sensitive short-circuit current in distal mouse colonic mucosa. This effect was absent in BK α-subunit-deficient (BK−/−) mice. The resting and diet-induced K+ secretion was stimulated by luminal ionomycin. In BK−/− mice luminal ionomycin did not stimulate K+ secretion. In vitro addition of aldosterone likewise triggered a 2-fold increase in K+ secretion, which was inhibited by the mineralocorticoid receptor antagonist spironolactone and the BK channel blocker IBTX. Semi-quantification of mRNA from colonic crypts showed up-regulation of BK α- and β2-subunits in high K+ diet mice. The BK channel could be detected luminally in colonic crypt cells by immunohistochemistry. The expression level of the channel in the luminal membrane was strongly up-regulated in K+-loaded animals. Taken together, these data strongly suggest that aldosterone-induced K+ secretion occurs via increased expression of luminal BK channels.

Mast cell stimulation by Ag is followed by the opening of Ca(2+)-activated K(+) channels, which participate in the orchestration of mast cell degranulation. The present study has been performed to explore the involvement of the Ca(2+)-activated K(+) channel K(Ca)3.1 in mast cell function. To this end mast cells have been isolated and cultured from the bone marrow (bone marrow-derived mast cells (BMMCs)) of K(Ca)3.1 knockout mice (K(Ca)3.1(-/-)) and their wild-type littermates (K(Ca)3.1(+/+)). Mast cell number as well as in vitro BMMC growth and CD117, CD34, and FcepsilonRI expression were similar in both genotypes, but regulatory cell volume decrease was impaired in K(Ca)3.1(-/-) BMMCs. Treatment of the cells with Ag, endothelin-1, or the Ca(2+) ionophore ionomycin was followed by stimulation of Ca(2+)-activated K(+) channels and cell membrane hyperpolarization in K(Ca)3.1(+/+), but not in K(Ca)3.1(-/-) BMMCs. Upon Ag stimulation, Ca(2+) entry but not Ca(2+) release from intracellular stores was markedly impaired in K(Ca)3.1(-/-) BMMCs. Similarly, Ca(2+) entry upon endothelin-1 stimulation was significantly reduced in K(Ca)3.1(-/-) cells. Ag-induced release of beta-hexosaminidase, an indicator of mast cell degranulation, was significantly smaller in K(Ca)3.1(-/-) BMMCs compared with K(Ca)3.1(+/+) BMMCs. Moreover, histamine release upon stimulation of BMMCs with endothelin-1 was reduced in K(Ca)3.1(-/-) cells. The in vivo Ag-induced decline in body temperature revealed that IgE-dependent anaphylaxis was again significantly (by approximately 50%) blunted in K(Ca)3.1(-/-) mice. In conclusion, K(Ca)3.1 is required for Ca(2+)-activated K(+) channel activity and Ca(2+)-dependent processes such as endothelin-1- or Ag-induced degranulation of mast cells, and may thus play a critical role in anaphylactic reactions.

17,18-Epoxyeicosatetraenoic acid (17,18-EETeTr) stimulates vascular large-conductance K(+) (BK) channels. BK channels are composed of the pore-forming BK alpha and auxiliary BK beta1 subunits that confer an increased sensitivity for changes in membrane potential and calcium to BK channels. Ryanodine-sensitive calcium-release channels (RyR3) in the sarcoplasmic reticulum (SR) control the process. To elucidate the mechanism of BK channel activation, we performed whole-cell and perforated-patch clamp experiments in freshly isolated cerebral and mesenteric artery vascular smooth muscle cells (VSMC) from Sprague-Dawley rats, BK beta1 gene-deficient (-/-), BK alpha (-/-), RyR3 (-/-) and wild-type mice. The 17,18-EETeTr (100 nm) increased tetraethylammonium (1 mm)-sensitive outward K(+) currents in VSMC from wild-type rats and wild-type mice. The effects were not inhibited by the epoxyeicosatrienoic acid (EET) antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (10 mum). BK channel currents were increased 3.5-fold in VSMC from BK beta1 (-/-) mice, whereas a 2.9-fold stimulation was observed in VSMC from RyR3 (-/-) mice (at membrane voltage 60 mV). The effects were similar compared with those observed in cells from wild-type mice. The BK current increase was neither influenced by strong internal calcium buffering (Ca(2)(+), 100 nm), nor by external calcium influx. The 17,18-EETeTr did not induce outward currents in VSMC BK alpha (-/-) cells. We next tested the vasodilator effects of 17,18-EETeTr on isolated arteries of BK alpha-deficient mice. Vasodilatation was largely inhibited in cerebral and mesenteric arteries isolated from BK alpha (-/-) mice compared with that observed in wild-type and BK beta1 (-/-) arteries. We conclude that 17,18-EETeTr represents an endogenous BK channel agonist and vasodilator. Since 17,18-EETeTr is active in small arteries lacking BK beta1, the data further suggest that BK alpha represents the molecular target for the principal action of 17,18-EETeTr. Finally, the action of 17,18-EETeTr is not mediated by changes of the internal global calcium concentration or local SR calcium release events.

Aim: Colonic crypts are the site of Cl - secretion. Basolateral K + channels provide the driving force for luminal cystic fibrosis transmembrane regulator-mediated Cl - exit. Relevant colonic epithelial K + channels are the intermediate conductance Ca 2+ -activated K ca 3.1 (SK4) channel and the cAMP-activated K v 7.1 (KCNQ1) channel. In addition, big conductance Ca 2+ -activated K ca 1.1 (BK) channels may play a role in Ca 2+ -activated Cl - secretion. Here we use K ca 1.1 and K ca 3.1 knock-out mice, and the K v 7.1 channel inhibitor 293B (10 μM) to investigate the role of K ca 1.1, K ca 3.1 and K v 7.1 channels in cholinergic-stimulated Cl - secretion. Methods: A Ussing chamber was used to quantify agonist-stimulated increases in short circuit current (I sc ) in distal colon. Chloride secretion was activated by bl. forskolin (FSK, 2 μM) followed by bl. carbachol (CCH, 100 μM). Luminal Ba 2+ (5 mM) was used to inhibit K ca 1.1 channels. Results: K ca 1.1 WT and KO mice displayed identical FSK and CCH-stimulated I sc changes, indicating that K ca 1.1 channels are not involved in FSK- and cholinergic-stimulated Cl - secretion. CCH-stimulated ∇I sc was significantly reduced in K ca 3.1 KO mice, underscoring the known relevance of this channel in the activation of Cl - secretion by an intracellular Ca 2+ increasing agonist. The residual CCH effect observed in K ca 3.1 KO mice suggests that yet another K + channel is driving the CCH-stimulated Cl - secretion. In the presence of the specific K v 7.1 channel blocker 293B, the residual CCH effect was abolished. Conclusions: This demonstrates that both K ca 3.1 and K v 7.1 channels are activated by cholinergic agonists and drive Cl - secretion. In contrast, K ca 1.1 channels are not involved in stimulated electrogenic Cl - secretion.

The unique voltage- and Ca2+-dependent K+ (BK) channel, prominently expressed in airway smooth muscle cells, has been suggested as an important effector in controlling airway contractility. Its deletion in mice depolarized resting membrane potential of tracheal cells, suggesting an increased open-probability of voltage-gated Ca2+ channels. While carbachol concentration-dependently increased the tonic tension of wild-type (WT) trachea, mutant trachea showed a different response with rapid tension development followed by phasic contractions superimposed on a tonic component. Tonic contractions were substantially more dependent on L-type Ca2+ current in mutant than in WT trachea, even though L-type Ca2+ channels were not up-regulated. In the absence of L-type Ca2+ current, half-maximal contraction of trachea was shifted from 0.51 to 1.7 microM. In agreement, cholinergic bronchoconstriction was reduced in mutant lung slices, isolated-perfused lungs and, most impressively, in mutant mice analyzed by body plethysmography. Furthermore, isoprenaline-mediated airway relaxation was enhanced in mutants. In-depth analysis of cAMP and cGMP signaling revealed up-regulation of the cGMP pathway in mutant tracheal muscle. Inhibition of cGMP kinase reestablished normal sensitivity toward carbachol, indicating that up-regulation of cGMP signaling counterbalances for BK channel ablation, pointing to a predominant role of BK channel in regulation of airway tone.

K(+) secretion in the kidney and distal colon is a main determinant of K(+) homeostasis. This study investigated the identity of the relevant luminal secretory K(+) ion channel in distal colon. An Ussing chamber was used to measure ion transport in the recently generated BK channel-deficient (BK(-/-)) mice. BK(-/-) mice display a significant colonic epithelial phenotype with (1) lack of Ba(2+)-sensitive resting K(+) secretion, (2) absence of K(+) secretion stimulated by luminal P2Y(2) and P2Y(4) receptors, (3) absence of luminal Ca(2+) ionophore (A23187)-stimulated K(+) secretion, (4) reduced K(+) and increased Na(+) contents in feces, and (5) an increased colonic Na(+) absorption. In contrast, resting and uridine triphosphate (UTP)-stimulated K(+) secretion was not altered in mice that were deficient for the intermediate conductance Ca(2+)-activated K(+) channel SK4. BK channels localize to the luminal membrane of crypt, and reverse transcription-PCR results confirm the expression of the BK channel alpha-subunit in isolated distal colonic crypts. It is concluded that BK channels are the responsible K(+) channels for resting and stimulated Ca(2+)-activated K(+) secretion in mouse distal colon.

The cGMP-dependent protein kinase type I (PKG I) is an essential regulator of cellular function in blood vessels throughout the body. DT-2, a peptidic inhibitor of PKG, has played a central role in determining the molecular mechanisms of vascular control involving PKG and its signaling partners. Here, we report the development of ( d )-amino acid DT-2 derivatives, namely the retro-inverso ri-( d )-DT-2 and the all ( d )-amino acid analog, ( d )-DT-2. Both peptide analogs were potent PKG Iα inhibitors with K i values of 5.5 nM (ri-( d )-DT-2) and 0.8 nM (( d )-DT-2) as determined using a hyperbolic mixed-type inhibition model. Also, both analogs were proteolytically stable in vivo , showed elevated selectivity, and displayed enhanced membrane translocation properties. Studies on isolated arteries from the resistance vasculature demonstrated that intraluminally perfused ( d )-DT-2 significantly inhibited vasodilation induced by 8-Br-cGMP. Furthermore, in vivo application of ( d )-DT-2 established a uniform translocation pattern in the resistance vasculature, with exception of the brain. Thus, ( d )-DT-2 caused significant increases in mean arterial blood pressure in unrestrained, awake mice. Further, mesenteric arteries isolated from ( d )-DT-2 treated animals showed a markedly reduced dilator response to 8-Br-cGMP in vitro . Our results clearly demonstrate that ( d )-DT-2 is a superior inhibitor of PKG Iα and its application in vivo leads to sustained inhibition of PKG in vascular smooth muscle cells. The discovery of ( d )-DT-2 may help our understanding of how blood vessels constrict and dilate and may also aid the development of new strategies and therapeutic agents targeted to the prevention and treatment of vascular disorders such as hypertension, stroke and coronary artery disease.

Oxygen-dependent antimicrobial activity of human polymorphonuclear leukocytes (PMNs) relies on the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to generate oxidants. As the oxidase transfers electrons from NADPH the membrane will depolarize and concomitantly terminate oxidase activity, unless there is charge translocation to compensate. Most experimental data implicate proton channels as the effectors of this charge compensation, although large-conductance Ca2+-activated K+ (BK) channels have been suggested to be essential for normal PMN antimicrobial activity. To test this latter notion, we directly assessed the role of BK channels in phagocyte function, including the NADPH oxidase. PMNs genetically lacking BK channels (BK−/−) had normal intracellular and extracellular NADPH oxidase activity in response to both receptor-independent and phagocytic challenges. Furthermore, NADPH oxidase activity of human PMNs and macrophages was normal after treatment with BK channel inhibitors. Although BK channel inhibitors suppressed endotoxin-mediated tumor necrosis factor-α secretion by bone marrow-derived macrophages (BMDMs), BMDMs of BK−/− and wild-type mice responded identically and exhibited the same ERK, PI3K/Akt, and nuclear factor-κB activation. Based on these data, we conclude that the BK channel is not required for NADPH oxidase activity in PMNs or macrophages or for endotoxin-triggered tumor necrosis factor-α release and signal transduction BMDMs.

Atherosclerotic vascular lesions are considered to be the predominant cause of morbidity and mortality in industrialized nations. Rupture of atherosclerotic plaques initiates platelet activation/aggregation with subsequent arterial thrombosis triggering myocardial infarction and stroke. Nitric oxide (NO) and its second messenger cyclic GMP (cGMP) comprise the central regulatory pathway that prevents platelet activation/aggregation under physiological conditions is. A major downstream target of NO is cyclic guanosine 3',5'-monophosphate kinase I (cGKI). In previous work, we have tested the intravascular significance of the NO/cGKI signalling pathway in vivo using cGKI-deficient (cGKI-/-) mice. We show that platelet cGKI but not endothelial or smooth muscle cGKI is essential to prevent intravascular adhesion and aggregation of platelets after ischemia. Correspondingly, loss of cGKI in platelets was associated with an increase in platelet accumulation in the postischemic kidney and with a significant enhancement of both platelet adhesion and aggregation in the postischemic intestinal microvasculature. The defect in platelet cGKI is not compensated by the cAMP/cAMP kinase pathway supporting the essential role of cGKI in prevention of ischemia-induced platelet adhesion and aggregation. Next, we addressed the regulatory cascade downstream of cGKI. We found that the inositol1,4,5-trisphosphate receptor-associated cGMP kinase substrate (IRAG) is abundantly expressed in platelets and assembled in a macrocomplex together with cGKI and the inositol-1,4,5-trisphosphate receptor type I (InsP3RI). cGKI phosphorylates IRAG at Ser664 and Ser677 in intact platelets. Targeted deletion of the IRAG-InsP3RI interaction in IRAGDelta12/Delta12 mutant mice leads to a loss of NO/cGMP-dependent inhibition of fibrinogen-receptor activation and platelet aggregation. Intracellular calcium transients were not affected by DEA/NO or cGMP in mutant platelets. Furthermore, intravital microscopy shows that NO fails to prevent arterial thrombosis of the injured carotid artery in IRAGDelta12/Delta12 mutants.

Address: 1Pharmazentrum Frankfurt/ZAFES, Institut fur Klinische Pharmakologie, Johann Wolfgang Goethe-Universitat, Frankfurt am Main, Germany, 2Pharmazeutisches Institut, Pharmakologie und Toxikologie, Tubingen, Germany, 3Institut fur Pharmakologie und Toxikologie, Technische Universitat, Munchen, Germany, 4Implen GmbH, Munchen, Germany and 5Zentrum fur Innere Medizin, Klinikum GarmischPartenkirchen, Garmisch-Partenkirchen, Germany

The kidney is the major regulator of potassium homeostasis. In addition to the ROMK channels, large conductance Ca(2+)-activated K(+) (BK) channels are expressed in the apical membrane of the aldosterone sensitive distal nephron where they could contribute to renal K(+) secretion. We studied flow-induced K(+) secretion in BK channel alpha-subunit knockout (BK(-/-)) mice by acute pharmacologic blockade of vasopressin V(2) receptors, which caused similar diuresis in wild-type and knockout mice. However, wild-type mice, unlike the BK(-/-), had a concomitant increase in urinary K(+) excretion and a significant correlation between urinary flow rate and K(+) excretion. Both genotypes excreted similar urinary amounts of K(+) irrespective of K(+) diet. This was associated, however, with higher plasma aldosterone and stronger expression of ROMK in the apical membrane of the aldosterone-sensitive portions of the distal nephron in the knockout than in the wild-type under control diet and even more so with the high-K(+) diet. High-K(+) intake significantly increased the renal expression of the BK channel in the wild-type mouse. Finally, despite the higher plasma K(+) and aldosterone levels, BK(-/-) mice restrict urinary K(+) excretion when placed on a low-K(+) diet to the same extent as the wild-type. These studies suggest a role of the BK channel alpha-subunit in flow-induced K(+) secretion and in K(+) homeostasis. Higher aldosterone and an upregulation of ROMK may compensate for the absence of functional BK channels.

Large-conductance Ca2+-activated K+(BK) channels are reported to be essential for NADPH oxidase-dependent microbial killing and innate immunity in leukocytes. Using human peripheral blood and mouse bone marrow neutrophils, pharmacological targeting, and BK channel gene-deficient (BK−/−) mice, we stimulated NADPH oxidase activity with 12- O-tetradecanoylphorbol-13-acetate (PMA) and performed patch-clamp recordings on isolated neutrophils. Although PMA stimulated NADPH oxidase activity as assessed by O2−and H2O2production, our patch-clamp experiments failed to show PMA-activated BK channel currents in neutrophils. In our studies, PMA induced slowly activating currents, which were insensitive to the BK channel inhibitor iberiotoxin. Instead, the currents were blocked by Zn2+, which indicates activation of proton channel currents. BK channels are gated by elevated intracellular Ca2+and membrane depolarization. We did not observe BK channel currents, even during extreme depolarization to +140 mV and after elevation of intracellular Ca2+by N-formyl-l-methionyl-l-leucyl-phenylalanine. As a control, we examined BK channel currents in cerebral and tibial artery smooth muscle cells, which showed characteristic BK channel current pharmacology. Iberiotoxin did not block killing of Staphylococcus aureus or Candida albicans. Moreover, we addressed the role of BK channels in a systemic S. aureus and Yersinia enterocolitica mouse infection model. After 3 and 5 days of infection, we found no differences in the number of bacteria in spleen and kidney between BK−/−and BK+/+mice. In conclusion, our experiments failed to identify functional BK channels in neutrophils. We therefore conclude that BK channels are not essential for innate immunity.

Large-conductance voltage- and Ca(2+)-activated K+ channels (BKCa) are involved in shaping spiking patterns in many neurons. Less is known about their role in mammalian inner hair cells (IHCs), mechanosensory cells with unusually large BKCa currents. These currents may be involved in shaping the receptor potential, implying crucial importance for the properties of afferent auditory signals. We addressed the function of BKCa by recording sound-induced responses of afferent auditory nerve (AN) fibers from mice with a targeted deletion of the pore-forming alpha-subunit of BKCa (BKalpha(-/-)) and comparing these with voltage responses of current-clamped IHCs. BKCa-mediated currents in IHCs were selectively abolished in BKalpha(-/-), whereas cochlear physiology was essentially normal with respect to cochlear sensitivity and frequency tuning.BKalpha(-/-) AN fibers showed deteriorated precision of spike timing, measured as an increased variance of first spike latency in response to tone bursts. This impairment could be explained by a slowed voltage response in the presynaptic IHC resulting from the reduced K+ conductance in the absence of BKCa. Maximum spike rates of AN fibers were reduced nearly twofold in BKalpha(-/-), contrasting with increased voltage responses of IHCs. In addition to presynaptic changes, which may be secondary to a modest depolarization of BKalpha(-/-) IHCs, this reduction in AN rates suggests a role of BKCa in postsynaptic AN neurons, which was supported by increased refractory periods. In summary, our results indicate an essential role of IHC BKCa channels for precise timing of high-frequency cochlear signaling as well as a function of BKCa in the primary afferent neuron.

cGMP-dependent protein kinase I (PKG-I) has been suggested to contribute to the facilitation of nociceptive transmission in the spinal cord presumably by acting as a downstream target of nitric oxide. However, PKG-I activators caused conflicting effects on nociceptive behavior. In the present study we used PKG-I -/- mice to further assess the role of PKG-I in nociception. PKG-I deficiency was associated with reduced nociceptive behavior in the formalin assay and zymosan-induced paw inflammation. However, acute thermal nociception in the hot-plate test was unaltered. After spinal delivery of the PKG inhibitor, Rp-8-Br-cGMPS, nociceptive behavior of PKG-I +/+ mice was indistinguishable from that of PKG-I -/- mice. On the other hand, the PKG activator, 8-Br-cGMP (250 nmol intrathecally) caused mechanical allodynia only in PKG-I +/+ mice, indicating that the presence of PKG-I was essential for this effect. Immunofluorescence studies of the spinal cord revealed additional morphological differences. In the dorsal horn of 3- to 4-week-old PKG-I -/- mice laminae I-III were smaller and contained fewer neurons than controls. Furthermore, the density of substance P-positive neurons and fibers was significantly reduced. The paucity of substance P in laminae I-III may contribute to the reduction of nociception in PKG-I -/- mice and suggests a role of PKG-I in substance P synthesis.

Abstract —Both cGMP-dependent and -independent mechanisms have been implicated in the regulation of vascular tone by NO. We analyzed acetylcholine (ACh)- and NO-induced relaxation in pressurized small arteries and aortic rings from wild-type (wt) and cGMP kinase I–deficient (cGKI –/– ) mice. Low concentrations of NO and ACh decreased the spontaneous myogenic tone in wt but not in cGKI –/– arteries. However, contractions of cGKI –/– arteries and aortic rings were reduced by high concentrations (10 μmol/L) of 2-( N , N -diethylamino)-diazenolate-2-oxide (DEA-NO). Iberiotoxin, a specific blocker of Ca 2+ -activated K + (BK Ca ) channels, only partially prevented the relaxation induced by DEA-NO or ACh in pressurized vessels and aortic rings. DEA-NO increased the activity of BK Ca channels only in vascular smooth muscle cells isolated from wt cGKI +/+ mice. These results suggest that low physiological concentrations of NO decrease vascular tone through activation of cGKI, whereas high concentrations of DEA-NO relax vascular smooth muscle independent of cGKI and BK Ca . NO-stimulated, cGKI-independent relaxation was antagonized by the inhibition of soluble guanylyl cyclase or cAMP kinase (cAK). DEA-NO increased cGMP to levels that are sufficient to activate cAK. cAMP-dependent relaxation was unperturbed in cGKI –/– vessels. In conclusion, low concentrations of NO relax vessels by activation of cGKI, whereas in the absence of cGKI, NO can relax small and large vessels by cGMP-dependent activation of cAK.

Atherosclerotic vascular lesions are considered to be a major cause of ischemic diseases, including myocardial infarction and stroke. Platelet adhesion and aggregation during ischemia–reperfusion are thought to be the initial steps leading to remodeling and reocclusion of the postischemic vasculature. Nitric oxide (NO) inhibits platelet aggregation and smooth muscle proliferation. A major downstream target of NO is cyclic guanosine 3′,5′-monophosphate kinase I (cGKI). To test the intravascular significance of the NO/cGKI signaling pathway in vivo, we have studied platelet–endothelial cell and platelet–platelet interactions during ischemia/reperfusion using cGKI-deficient (cGKI−/−) mice. Platelet cGKI but not endothelial or smooth muscle cGKI is essential to prevent intravascular adhesion and aggregation of platelets after ischemia. The defect in platelet cGKI is not compensated by the cAMP/cAMP kinase pathway supporting the essential role of cGKI in prevention of ischemia-induced platelet adhesion and aggregation.

Regulation of smooth muscle contractility is essential for many important biological processes such as tissue perfusion, cardiovascular haemostasis and gastrointestinal motility. While an increase in calcium initiates smooth muscle contraction, relaxation can be induced by cGMP or cAMP. cGMP-dependent protein kinase I (cGKI) has been suggested as a major mediator of the relaxant effects of both nucleotides. To study the biological role of cGKI and its postulated cross-activation by cAMP, we inactivated the gene coding for cGKI in mice. Loss of cGKI abolishes nitric oxide (NO)/cGMP-dependent relaxation of smooth muscle, resulting in severe vascular and intestinal dysfunctions. However, cGKI-deficient smooth muscle responded normally to cAMP, indicating that cAMP and cGMP signal via independent pathways, with cGKI being the specific mediator of the NO/cGMP effects in murine smooth muscle.