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Neutrophil migration into infected tissues is essential for host defense, but products of activated neutrophils can be quite damaging to host cells. Neutrophil influx into the lung and airways and resultant inflammation characterizes diseases such as chronic obstructive pulmonary disease, bronchiectasis, and cystic fibrosis. To migrate, neutrophils must reorganize the actin cytoskeleton to establish a leading edge pseudopod and a trailing edge uropod. The actin-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) has been shown to bind and cross-link actin in a variety of cell types and to co-localize with F-actin in the leading edge lamellipodium of migrating fibroblasts. The hypothesis that MARCKS has a role in the regulation of neutrophil migration was tested using a cell-permeant peptide derived from the MARCKS myristoylated aminoterminus (MANS peptide). Treatment of isolated human neutrophils with MANS significantly inhibited both their migration and beta2 integrin-dependent adhesion in response to N-formyl-methionyl-leucyl-phenylalanine (fMLF), IL-8, or leukotriene (LT)B(4). The IC(50) for fMLF-induced migration and adhesion was 17.1 microM and 12.5 microM, respectively. MANS significantly reduced the F-actin content in neutrophils 30 seconds after fMLF stimulation, although the peptide did not alter the ability of cells to polarize or spread. MANS did not alter fMLF-induced increases in surface beta2 integrin expression. These results suggest that MARCKS, via its myristoylated aminoterminus, is a key regulator of neutrophil migration and adhesion.

Equine laminar tissues do not contain resident neutrophils and have less superoxide dismutase (SOD) activity than other equine tissues, which makes them inherently more vulnerable to damage induced by reactive oxygen species (ROS) produced by neutrophils that enter the tissues. In the advanced clinical stages of acute laminitis, pathologic events in affected feet include a breakdown in the basement membrane, neutrophil infiltration, and platelet-neutrophil aggregates in laminar dermal veins, highlighting the contribution of neutrophils to the pathophysiology of the disease. The aim of this study was to determine the role of p38 MAPK in the mechanism underlying equine neutrophil migration to potentially reveal therapeutic targets that may limit lamellar damage from the neutrophil influx that occurs in acute laminitis. We determined that the endogenous chemoattractant LTB(4) transiently activated p38 MAPK and induced chemotaxis of equine primary neutrophils. Inhibition with the p38 MAPK specific inhibitor SB203580 reduced LTB(4)-induced migration in a dose-dependent manner with an IC(50) of 2.8 microM. We then examined the potential mechanisms underlying the ability of SB203580 to abolish migration. We determined that inhibition of p38 MAPK with 10 microM SB203580 disrupted the ability of neutrophils to polarize in response to LTB(4) and PAF. In contrast, p38 MAPK did not appear to be required for chemoattractant- or PKC-induced beta2 integrin-dependent adhesion or chemoattractant-induced upregulation of surface beta2 integrins, but was required for TNFalpha-induced adhesion. These findings support a function for p38 MAPK in equine neutrophil migration and suggest the potential for the ability of p38 MAPK inhibition to limit neutrophilic inflammation in the laminae during acute laminitis.

Endotoxemia occurs when bacterial lipopolysaccharide (LPS) in the blood induces a dysregulated inflammatory response, resulting in circulatory shock and multi-organ failure. Laminitis is a common complication in endotoxemic horses and is frequently the reason for humane euthanasia of these cases. Blood leukocytes are a principal target of LPS in endotoxemia leading to activation of multiple signal transduction pathways involved in the induction of a number of pro-inflammatory genes. In other animal models, the p38 mitogen activated protein kinase (MAPK) pathway has been associated with induced expression of tumor necrosis factor-alpha (TNFalpha), interleukin (IL)-1beta, IL-6 and IL-8. The goal of this study was to determine the role of the p38 MAPK pathway in the induction of these pro-inflammatory cytokine and chemokine genes in LPS-stimulated equine leukocytes. Stimulation of equine peripheral blood leukocytes resulted in an increase in TNFalpha, IL-1beta, IL-6 and IL-8 mRNA levels. Pharmacological inhibition of p38 MAPK activity with SB203580 or SB202190 reduced the ability of LPS stimulation to increase mRNA concentrations for all four genes. However, only SB203580 pretreatment significantly reduced LPS-stimulated IL-1beta and IL-8 mRNA expression and only pretreatment with SB202190 significantly reduced LPS-stimulated TNFalpha and IL-6 mRNA expression. From this study we conclude TNFalpha, IL-1beta, IL-6 and IL-8 are induced upon LPS stimulation of equine leukocytes and that this induction of gene expression is dependent on the p38 MAPK pathway. However, there are differences in the efficacy of the p38 inhibitors tested here that may be explained by differences in specificity or potency. This study provides evidence for the use of selective p38 MAPK inhibitors as potential therapeutics for the treatment of equine endotoxemia.

Vasodilator-stimulated phosphoprotein (VASP) is a cAMP-dependent protein kinase A (PKA) substrate, which links cellular signaling to cytoskeletal organization and cellular movement. VASP is phosphorylated by PKA on serine 157 (Ser 157), which is required for VASP function in platelet adhesion and fibroblast motility. Our hypothesis is that PKA regulates neutrophil migration through VASP Ser 157 phosphorylation. The objective of this study was to characterize VASP Ser 157 phosphorylation in chemoattractant-stimulated neutrophils. fMLF, IL-8, leukotriene B(4), or platelet-activating factor stimulation resulted in an initial increase in VASP Ser 157 phosphorylation, which was maximal by 30 s and was followed by a return to baseline Ser 157 phosphorylation by 10 min. In contrast, stimulation with the nonchemoattractant, proinflammatory cytokine TNF-alpha did not affect Ser 157 phosphorylation. The kinetics of fMLF-induced VASP Ser 157 phosphorylation levels closely matched the kinetics of the fold-change in F-actin levels in fMLF-stimulated neutrophils. fMLF-induced Ser 157 phosphorylation was abolished by pretreatment with the PKA inhibitor H89 and the adenylyl cyclase inhibitor SQ22536. In contrast, fMLF-induced Ser 157 phosphorylation was unaffected by the PKC inhibitors calphostin and staurosporine, the PKG inhibitors Rp-8-pCPT-cGMP and KT5823, and the calmodulin-dependent protein kinase II inhibitor KN-62. Inhibition of adhesion with EDTA or the anti-beta2-integrin antibody IB4 did not alter fMLF-induced VASP phosphorylation or dephosphorylation. These data show that chemoattractant stimulation of human neutrophils induces a rapid and transient PKA-dependent VASP Ser 157 phosphorylation. Adhesion does not appear to be an important regulator of the state of VASP Ser 157 phosphorylation in chemoattractant-stimulated neutrophils.

The goal of this study was to define the role for p38 mitogen-activated kinase (MAPK) in the signaling mechanism regulating pro-inflammatory cyclooxygenase (COX) gene expression in lipopolysaccharide (LPS)-activated equine leukocytes for the purposes of identifying novel targets for anti-inflammatory therapy in endotoxemic horses. The p38 MAPK has been shown to positively regulate inflammatory gene expression in human leukocytes and can be activated by a variety of stimuli including LPS, TNF-alpha, and IL-1. Activation-associated phosphorylated p38 MAPK has been implicated in the up-regulation of several inflammatory genes, including COX-2 which ultimately results in the production of prostanoids that are responsible for the pathophysiology associated with endotoxemia. Our hypothesis is that activation of p38 MAPK is essential for LPS-induced COX-2 expression in equine peripheral blood leukocytes. We tested our hypothesis by investigating the effects of the specific p38 MAPK inhibitors SB203580 and SB202190 on LPS-induced COX-2 protein expression and PGE(2) production in equine leukocytes. LPS stimulation activated p38 MAPK and increased COX-2 expression in a dose-dependent manner with maximal activation observed after 30min and 4h, respectively, at a concentration of 10 ng/ml LPS. In contrast, LPS stimulation did not affect COX-1 protein expression. Pretreatment with SB203580 or SB202190 significantly inhibited LPS-induced activation-associated p38 MAPK phosphorylation, COX-2 mRNA and protein levels, and PGE(2) production in equine leukocytes. Maximal inhibition of LPS-induced COX-2 protein expression was achieved at a concentration of 10 microM SB203580. We concluded that p38 MAPK is essential for LPS-induced COX-2 expression suggesting that p38 MAPK is a potential target for anti-inflammatory therapy during equine endotoxemia.

Objective: To determine the participation of GTP-binding proteins (G-proteins) in the cellular mechanism of the phenylephrine-induced renal artery vasospasm by using swine renal artery smooth muscle rings in a standard organ baths, as increased noradrenaline release from perivasal and intramural sympathetic nerve endings during renal ischaemia results in increased vascular smooth muscle tone that is important in the loss of kidney function during renal transplantation and nephron-sparing surgery., Materials and Methods: Fresh swine kidneys were transported in cold calcium-free Tyrode solution to the laboratory. Adipose tissue around the arteries was removed, the organ de-capsulated and interlobar arteries dissected. The contractile properties of renal artery smooth muscle rings were assessed in a standard organ bath, the rings pre-tensioned at 2 g. Contractions were evoked by applying the alpha 1-adrenoceptor selective agonist phenylephrine (1 nmol/L to 0.3 mmol/L). Isometric contractions of the tissue were registered and stored digitally. Dose-response curves were obtained sequentially with a wash-out of 20 min between each concentration; the maximum contractility of an individual muscle ring was set at 100%. Dose-response curves of inhibitory agents (e.g. WB4101, cholera and pertussis toxins) were determined by comparing the remaining contractility after incubating with the respective drug with a control contraction that was evoked three times (10 mumol/L phenylephrine) and the mean set at 100%., Results: Phenylephrine induced dose-dependent and fully reversible isometric contractions with a threshold concentration of 100 nmol/L and an EC50 of 0.8 mumol/L. The receptor was identified as the alpha 1A-subtype by the selective antagonist WB4101. Pre-treatment of tissue rings with 5 micrograms/mL pertussis toxin (120 min, 37 degrees C) inhibited the control contraction by a mean (SEM) of 52.0 (4.6)%, whereas pre-treatment with 1 microgram/mL cholera toxin (60 min, 37 degrees C), leading to a permanent activation of the Gs-protein via blockade of the GTPase activity, decreased the response by 39.0 (8.2%)., Conclusion: These results suggest a coupling of alpha 1A-adrenoceptors in renal vascular tissue to the heterotrimeric Gs-protein and to heterotrimeric G-proteins of the G1- and/or G0-family in the phenylephrine-induced contraction.

The elevation of vascular smooth muscle tone in the renal arteries during kidney transplantation and nephron-sparing surgery plays a major role in postsurgical organ dysfunction. Therefore, a better understanding of the intracellular mechanisms of contraction and relaxation is of fundamental interest to improve urological treatment. The present study was designed to investigate the complex intracellular system of cyclic nucleotides involved in the regulation of smooth muscle relaxation by using swine renal artery rings in the Schuler organ bath. Phenylephrine (PE) induced dose-dependent and fully reversible isometric contractions with a threshold concentration of 10 nM and an EC(50) of 804 nM. The receptor was identified as alpha(1A)-subtype by the selective antagonist WB4101. Increasing the intracellular concentration of cyclic 3':5'-adenosine monophosphate (cAMP) by dibutyryl-cAMP (5 mM) and forskolin (5 micro M) resulted in a decreased contractiltity of 48.0% and 76.3%, respectively. Elevation of the cytosolic content of cyclic 3':5'-guanosine monophosphate (cGMP) using dibutyryl-cGMP (1 mM), sodium nitroprusside (100 micro M) and SIN-1 (100 micro M) decreased the average PE-induced contraction by 16.4%, 41.9% and 62.4%, respectively. The unselective phosphodiesterase inhibitors theophylline (1 mM), papaverine (100 micro M) and IBMX (5 mM) reduced the PE-induced contraction by 37.3%, 93.1% and 95.5%, respectively. Furthermore, selective inhibition of phosphodiesterases by milrinone (PDE(3)-selective) resulted in a decreased contractility by 1.3% (50 micro M), 29.5% (100 micro M) and 93.5% (5 mM), and using rolipram (PDE(4) selective), the PE-induced contraction was inhibited by 57.9% (50 micro M) and 81.9% (100 micro M). The results suggest the involvement of cAMP and cGMP in the relaxation of renal artery smooth muscle cells. Moreover, phosphodiesterases, especially PDE(3) and PDE(4), seem to play a critical role in the regulation of renal artery smooth muscle tone.

The ischemia induced vasospasm of the renal arterial blood vessels mediated by alpha1-adrenoceptors is of importance for the loss of kidney function. This is based on reduced perfusion of the kidney cortex occurring in kidney transplant and organ preserving surgery. The present study considered the intracellular mechanism of the norepinephrine (NE) induced renal artery vasospasm by using swine renal artery smooth muscle ring. Norepinephrine and phenylephrine (PE) induced dose-dependent and fully reversible isometric contractions with a threshold concentration of 10 nM (n = 7) and 10 nM (n = 4), and an EC50 of 0.3 microM and 1 microM, respectively. The receptor was identified as alpha1A-subtype. The contraction was completely inhibited by verapamil (IC50 = 1.51 microM; n = 11) and diltiazem (IC50 = 9.49 microM; n = 8) and 85% by nifedipine (IC50 = 0.13 microM; n = 21). Blockade of the intracellular inositol- 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store by thapsigargin (1 microM, n = 7) or suppression of Ca2+ release from the intracellular Ca2+-sensitive Ca2+ store by ryanodine (100 microM, n = 4) inhibited the PE induced contraction by 39.5% and 47.6%, respectively. The results suggest a key role of voltage-dependent Ca2+ channels and intracellular Ca2+ stores in the alpha1A-adrenoceptor induced contraction of the renal artery.

Objective: The aim of the present study was to evaluate the therapeutic potency of an electrotherapy of striated ischiocavernous muscles in patients with erectile dysfunction., Patients and Methods: Transcutaneous electrostimulation of striated ischiocavernous muscles by self-adhesive penile or perineal skin electrodes was performed in 48 patients with erectile dysfunction. 6/48 patients (R) responded to intracavernous pharmacotherapy while 42/48 (NR) did not show significant penile rigidity even to intracavernous papaverine/phentolamine/PGE1 triple drug medication., Results: Within the observation time of 3 months, 10/48 patients dropped out. 22/38 patients reported a penile rigidity for sufficient sexual intercourse whereby 3/22 NR required additional intracavernous pharmacotherapy. Penile rigidity could be objectivated by triple drug medicaton in 12/14 NR after ischiocavernous muscle stimulation (EIS) therapy. 5/6 R were treated successfully for premature erection loss. During EIS treatment neither discomfort nor complications could be observed., Conclusion: Transcutaneous electrostimulation of ischiocavernous muscles is a new, noninvasive therapy for the improvement of penile rigidity. The clinical results underline the importance of the striated ischiocavernous muscles for penile rigidity.

The increased sympathetic neurotransmission in benign prostatic hyperplasia (BPH) results in a alpha 1C-adrenoceptor-mediated increase in prostatic smooth muscle tone which seems to be responsible for the dynamic infravesical obstruction occurring in BPH. The prostatic smooth muscle contractions evoked by norepinephrine can be efficiently blocked by alpha 1-adrenoceptor blockers. Moreover, an impressive number of clinical trials illustrated the beneficial results of alpha 1-adrenoceptor blockers in the treatment of BPH. However, despite knowledge of alpha 1-adrenergic neurotransmission and the clinical application of its blockade by selective alpha 1-adrenoceptor antagonists, very little is known about the intracellular pathways involved in the regulation of prostatic smooth muscle contractility. To study the intracellular mechanism of the alpha 1C-adrenoceptor-induced prostatic smooth muscle contraction, the patch-clamp technique in the whole-cell configuration mode combined with the Fura-II fluorescence technique was used in human, enzymatically isolated smooth muscle cells obtained from patients undergoing transurethral resection of the prostate because of symptomatic BPH. Furthermore changes in prostatic smooth muscle contractility were registered in organ bath experiments. Application of the selective alpha 1-adrenoceptor agonist phenylephrine (PE) increased the L-type Ca(2+)-channel current (ICa) dose dependently from 8 up to 18.5 microA/cm2, simultaneously elevating the free cytoplasmic Ca2+ concentration ([Ca2+]i) up to 1.9 microM. Pretreating the myocytes with pertussis toxin, an exotoxin of Bordetella pertussis which inactivates GTP-binding proteins (G proteins) of the Gi and G(o) family by ADP ribosylation, reduced the PE-induced ICa stimulation by 71.5 +/- 5.6% (n = 21). Dialysis of the cytosol with the second messenger inositol-1,4,5-trisphosphate (IP3), which releases Ca2+ from intracellular non-mitochondrial, IP3-sensitive Ca2+ pools, imitated the PE-evoked responses. Pretreating the myocytes with the Ca(2+)-release blockers ryanodine (10-100 microM, n = 8), thapsigargin (0.1 microM, n = 11) or low-molecular weight heparin (n = 14) largely attenuated the PE-evoked responses. The experimental results suggest a coupling of alpha 1-adrenoceptors to phospholipase C-converting phosphoinositol-4,5-bisphosphate into diacylglycerol, an endogenous activator of the protein kinase C and IP3 which releases Ca2+ from intracellular stores stimulating ICa via Ca(2+)-calmodulin-dependent protein kinase induced phosphorylation of voltage-dependent Ca2+ channels. This knowledge could be of interest for conservative treatment in symptomatic BPH.

The role of cyclic 3'-5' adenosine monophosphate (cAMP) on alpha 1-adrenoceptor (alpha 1-receptor) induced smooth muscle contractions in symptomatic benign prostatic hyperplasia (BPH) was investigated. Application of the selective alpha 1-receptor agonist phenylephrine (PE) induced fully reversible contractions in a dose-dependent fashion. Phosphodiesterase (PDE) inhibitors blocking the degradation of cAMP suppressed the PE induced contractions as follows: theophylline (1 mM), 91.1 +/- 1.4%; papaverine (0.5 mM), 822.8 +/- 3.2%; milrinone (0.5 mM), 68.2 +/- 0.6%. Forskolin (50 microM), which elevates cAMP through direct activation of adenylatecyclase (AC), inhibited the PE induced contractions by 82.4 +/- 3.6%. To further increase the intracellular cAMP concentration ([cAMP]i), the membrane permeable cAMP analogue N6-2'-O-dibutyryladenosine derivative (dBcAMP; 1 mM) was applied and reduced the PE evoked contractions by 69.8 +/- 2.3%. We conclude that elevation of [cAMP]i is an important step in inducing smooth muscle relaxation.

Symptoms in benign prostatic hyperplasia (BPH) are partially caused by an increased prostatic smooth muscle tone. This tone depends on extra- and intracellular Ca2+ stores and is regulated by various second messenger pathways. We investigated the role of intracellular Ca2+ stores and cyclic 3'-5' adenosine monophosphate (cAMP) in BPH. Contractions elicited by the specific alpha 1-receptor agonist phenylephrine (PE) were inhibited by the selective alpha 1-receptor antagonists prazosin and YM 617. To elucidate the contribution of intracellular Ca2+ stores to the alpha 1-receptor induced contraction nifedipine, a blocker of voltage dependent L-type Ca2+ channels (VDCC) was applied and found to inhibit the PE induced contractions up to 65%. To further confirm the participation of intracellular Ca2+ stores, we applied ryanodine (10 microM) which reduced the alpha 1-receptor mediated contractions up to 80%. The remaining contraction was sensitive to nifedipine. The cAMP pathway mediating smooth muscle relaxation by regulating intracellular Ca2+ concentrations ([Ca2+]i) was also investigated. Nonspecific phosphodiesterase (PDE) inhibitors such as papaverine (0.5 mM) and theophylline (1 mM) and the specific PDE inhibitor milrinone (0.5 mM), all of which prevent degradation of cAMP, suppressed the PE induced contractions by 82%, 91% and 68%, respectively. Forskolin (50 microM), an activator of adenylylecyclase (AC), inhibited the PE induced contractions by 83%. The membrane permeable cAMP analog, N6-2'-0-dibutyryladenosine derivative (dBcAMP) also reduced the PE induced response by 70%.

The role of signal transducing guanine-nucleotide binding proteins (G-proteins) in alpha 1-receptor mediated smooth muscle contractions was investigated in human hyperplastic prostatic tissue. The selective alpha 1-receptor agonist phenylephrine (PE) evoked dose dependent contractions antagonized by the alpha 1-receptor blockers prazosin (EC50 10 nM) and YM617 (EC50 3 nM). Application of nifedipine (1-10,000 nM), a blocker of voltage-dependent L-type Ca(2+)-channels (VDCC), inhibited the PE evoked contraction up to 65.4%. Pretreating the tissue strips with pertussis toxin (PTX, exotoxin from Bordetella pertussis; 5-25 micrograms/ml), inactivating a subpopulation of G-proteins, inhibited the PE induced contractions up to 73.9%. PTX pretreatment had no effect on contractions elicited by 125 mM K+. Application of nifedipine to PTX pretreated tissue led to an additional inhibition of 13.7%. Our findings demonstrate the involvement of PTX-sensitive G-proteins in the signal transduction pathway of alpha 1-receptor induced contractions of prostatic smooth muscle. The remaining contractility of PTX pretreated tissue suggests additional participation of PTX insensitive mechanisms in alpha 1-receptor mediated prostatic smooth muscle contractions.

Guinea pig vas deferens was used as an animal model for alpha-1 adrenoceptor (alpha 1-receptor) mediated contractions in human hyperplastic prostatic tissue. The selective alpha 1-receptor agonist, phenylephrine (PE), induced fully reversible, dose-dependent contractions antagonized by increasing concentrations of the alpha 1-receptor blockers prazosin (1-100 nM) and YM 617 (0.1-10 nM). Removal of extracellular Ca2+ reduced PE-evoked contractions in a time-dependent manner. Nifedipine (1-1000 nM), a blocker of voltage-dependent L-type Ca2+ channels (VDCC), inhibited the PE-induced response by up to 65%. Removal of extracellular Ca2+ abolished the alpha 1-agonist reactivity in a time-dependent fashion. To elucidate the participation of intracellular Ca2+ stores in alpha 1-receptor-mediated contractions, the tissue was pretreated with ryanodine (10 microM) or thapsigargin (0.1 microM), established inhibitors of Ca2+ release from intracellular pools. Both substances reduced the PE contractions by up to 80%. Nifedipine suppressed the remaining contractions completely. This provides evidence that Ca2+ influx through VDCC and Ca2+ release from intracellular stores contribute to alpha 1-receptor-mediated contractions in the guinea pig vas deferens and may be important in obstructive benign prostatic hyperplasia.