Robert S. Moreland

Inhibition of p42 and p44 MAP kinase does not alter smooth muscle contraction in swine carotid artery

Caldesmon inhibits myosin ATPase activity; phosphorylation of caldesmon reverses the inhibition. The caldesmon kinase is believed to be mitogen-activated protein (MAP) kinase. MAP kinases are activated during vascular stimulation, but a cause-and-effect relationship between kinase activity and contraction has not been established. We examined the role of MAP kinase in contraction using PD-098059, an inhibitor of MAP kinase kinase (MEK). MAP kinase activity was assessed using an anti-active MAP kinase antibody and direct measurement of MAP kinase catalyzed phosphorylation of myelin basic protein, MBP-(95-98). MAP kinase phosphorylation, stimulated by histamine (50 microM) or phorbol 12,13-dibutyrate (PDBu, 0.1 microM), was inhibited by PD-098059 (100 microM). PD-098059 did not alter the sensitivity or the maximal level of force in smooth muscle stimulated by histamine or PDBu, nor did PD-098059 affect contraction of beta-escin-permeabilized tissue. Our data suggest that p44 and p42 MAP kinases are not involved in regulation of vascular smooth muscle contraction. These results do not, however, preclude a role for other isoforms of the MAP kinase family.Caldesmon inhibits myosin ATPase activity; phosphorylation of caldesmon reverses the inhibition. The caldesmon kinase is believed to be mitogen-activated protein (MAP) kinase. MAP kinases are activated during vascular stimulation, but a cause-and-effect relationship between kinase activity and contraction has not been established. We examined the role of MAP kinase in contraction using PD-098059, an inhibitor of MAP kinase kinase (MEK). MAP kinase activity was assessed using an anti-active MAP kinase antibody and direct measurement of MAP kinase catalyzed phosphorylation of myelin basic protein, MBP-(95-98). MAP kinase phosphorylation, stimulated by histamine (50 μM) or phorbol 12,13-dibutyrate (PDBu, 0.1 μM), was inhibited by PD-098059 (100 μM). PD-098059 did not alter the sensitivity or the maximal level of force in smooth muscle stimulated by histamine or PDBu, nor did PD-098059 affect contraction of β-escin-permeabilized tissue. Our data suggest that p44 and p42 MAP kinases are not involved in regulation of vascular smooth muscle contraction. These results do not, however, preclude a role for other isoforms of the MAP kinase family.

N1-Alkylated 3,4-dihydropyrimidine-2(1H)-ones: Convenient one-pot selective synthesis and evaluation of their calcium channel blocking activity

It has been found that selective N1-alkylation of 3,4-dihydropyrimidine-2(1H)-ones can be achieved under solvent-less, mild phase transfer catalytic (PTC) conditions with tetrabutylammonium hydrogen sulfate and 50% aqueous NaOH as the catalyst and base, respectively. The procedure is tolerant to substitutional variation at key diversity points on the pyrimidinone moiety.

Ca2+ sensitization pathways accessed by cholinergic neurotransmission in the murine gastric fundus

•  In smooth muscles, the sensitivity of contraction to Ca2+ can be increased by the phosphorylation of CPI‐17 and MYPT1, resulting in the inhibition of myosin light chain phosphatase (MLCP). •  Ca2+ sensitization of smooth muscle contraction has typically been studied by immersing muscles in solutions containing contractile agonists. •  However, stimulating muscles by bath‐applied agonists may not be equivalent to neurotransmitter release because different post‐junctional receptors may be activated in response to these different modes of stimulation. •  In this study we found that a bath‐applied cholinergic agonist activates Ca2+ sensitization mechanisms in gastric fundus smooth muscles that are different than those of cholinergic neurotransmission. Electrical field stimulation (EFS) only increased CPI‐17 phosphorylation, while bath‐applied carbachol increased both CPI‐17 and MYPT1 phosphorylation. •  With the cholinesterase inhibitor neostigmine present, both CPI‐17 and MYPT1 phosphorylation were increased by EFS. •  In fundus muscles of W/Wv mice which lack intramuscular interstitial cells of Cajal (ICC‐IMs), EFS alone increased both CPI‐17 and MYPT1 phosphorylation. •  These findings indicate that ACh availability determines which Ca2+ sensitization mechanisms are activated, and ICC‐IMs regulate the access of ACh to smooth muscles.

Rate-limiting energy-dependent steps controlling oxidative metabolism-contraction coupling in rabbit aorta.

1. We tested the hypotheses that coupling between oxidative metabolism and force in noradrenaline (NOR)‐activated rabbit aorta is controlled (a) by an energy‐dependent step or steps in receptor‐operated coupling mechanisms upstream to myosin light chain (MLC) kinase, or (b) by energy limitation of MLC kinase‐mediated phosphorylation of the MLC or actin‐activated myosin ATPase. 2. Oxidative energy production was rapidly inhibited by decreasing organ bath PO2 to less than 30 mmHg. There was no difference, comparing KCl‐ or NOR‐induced force, in the rates of decrease of [PCr] (phosphocreatine) or [ATP] following inhibition of oxidative energy production. (In this report we use the term [PCr] and [ATP] to indicate mean tissue values). Initial rates of decrease in [PCr] and [ATP] following inhibition of oxidative energy production were 0.05 mM/min and 0.06 mM/min, respectively. 3. Despite similar decreases in mean tissue [PCr] and [ATP], relaxations of KCl‐induced contractions following inhibition of oxidative energy production were markedly delayed and were blunted compared to relaxations seen during NOR‐induced contractions. The threshold mean tissue [PCr] and [ATP] for relaxation during KCl stimulation were 0.25 and 0.60 to 0.80 mM, respectively. During NOR stimulation, threshold values of [PCr] and [ATP] were 0.50 mM and 0.80 mM, respectively. Mean tissue [PCr] and [ATP] levels at 50% relaxation of KCl‐induced force were less than 0.1 mM and 0.1 mM, respectively. Fifty per cent relaxation of NOR‐induced force occurred at [PCr] and [ATP] values of 0.35 mM and 0.65 mM, respectively. 4. MLC phosphorylation levels decreased during relaxation of NOR force evoked by inhibition of oxidative energy production. There was no change in the level of MLC phosphorylation following inhibition of oxidative energy production in KCl‐contracted muscle even at mean tissue [PCr] and [ATP] lower than values associated with decreases in MLC phosphorylation during relaxations of NOR‐induced force. 5. Oxygen‐induced redevelopment of force during NOR exposure was not dependent on extracellular Ca2+. Mean tissue [PCr] increased prior to onset of O2‐evoked force redevelopment. Increases in MLC phosphorylation were seen at the time of onset of force redevelopment. 6. Oxidative metabolism‐contraction coupling during NOR‐stimulation seems not to be due to energy limitation of the MLC kinase reaction or actin‐activated myosin ATPase. Data suggest the rate‐limiting step is an energy‐dependent reaction in receptor‐operated coupling mechanisms upstream to MLC kinase.(ABSTRACT TRUNCATED AT 400 WORDS)

Carbachol-induced rabbit bladder smooth muscle contraction: roles of protein kinase C and Rho kinase

Smooth muscle contraction is regulated by phosphorylation of the myosin light chain (MLC) catalyzed by MLC kinase and dephosphorylation catalyzed by MLC phosphatase. Agonist stimulation of smooth muscle results in the inhibition of MLC phosphatase activity and a net increase in MLC phosphorylation and therefore force. The two pathways believed to be primarily important for inhibition of MLC phosphatase activity are protein kinase C (PKC)-catalyzed CPI-17 phosphorylation and Rho kinase (ROCK)-catalyzed myosin phosphatase-targeting subunit (MYPT1) phosphorylation. The goal of this study was to determine the roles of PKC and ROCK and their downstream effectors in regulating MLC phosphorylation levels and force during the phasic and sustained phases of carbachol-stimulated contraction in intact bladder smooth muscle. These studies were performed in the presence and absence of the PKC inhibitor bisindolylmaleimide-1 (Bis) or the ROCK inhibitor H-1152. Phosphorylation levels of Thr(38)-CPI-17 and Thr(696)/Thr(850)-MYPT1 were measured at different times during carbachol stimulation using site-specific antibodies. Thr(38)-CPI-17 phosphorylation increased concurrently with carbachol-stimulated force generation. This increase was reduced by inhibition of PKC during the entire contraction but was only reduced by ROCK inhibition during the sustained phase of contraction. MYPT1 showed high basal phosphorylation levels at both sites; however, only Thr(850) phosphorylation increased with carbachol stimulation; the increase was abolished by the inhibition of either ROCK or PKC. Our results suggest that during agonist stimulation, PKC regulates MLC phosphatase activity through phosphorylation of CPI-17. In contrast, ROCK phosphorylates both Thr(850)-MYPT1 and CPI-17, possibly through cross talk with a PKC pathway, but is only significant during the sustained phase of contraction. Last, our results demonstrate that there is a constitutively activate pool of ROCK that phosphorylates MYPT1 in the basal state, which may account for the high resting levels of MLC phosphorylation measured in rabbit bladder smooth muscle.

An Efficacious Protocol for 4‐Substituted 3,4‐Dihydropyrimidinones: Synthesis and Calcium Channel Binding Studies

Ethyl 1,2-dihydro-1,6-dimethyl/6-methyl-2-oxopyrimidine-5-carboxylates react with C-nucleophiles as well as anion of enantiopure chiral auxiliary (1R,2S,5R)-(-)-methyl (S)-p-toluenesulfinate to afford C-4 substituted and enantiopure congeners of medicinally potent Biginelli dihydropyrimidinones. The calcium channel blocking activity of some of the compounds was evaluated and compared with nifedipine for their ability to relax a membrane depolarization induced contraction.

Caldesmon phosphorylation is catalyzed by two kinases in permeabilized and intact vascular smooth muscle

Smooth muscle contraction is initiated by myosin light chain (MLC) phosphorylation catalyzed by the Ca2+ dependent MLC kinase. However, many aspects of smooth muscle contraction cannot be accounted for by MLC phosphorylation. One hypothesis that has received experimental support involves the thin filament protein caldesmon. Caldesmon inhibits myosin ATPase activity; phosphorylation of caldesmon relieves this inhibitory effect. The primary candidates for catalysis of caldesmon phosphorylation are the p42/p44 ERK MAP kinases. However, we and others have shown that inhibition of the ERK MAP kinases has no effect on many smooth muscles. The goal of this study was to determine if evidence for a second endogenous caldesmon kinase may be obtained. We used Triton X‐100 skinned and intact tissues of the swine carotid artery to address this goal. Caldesmon phosphorylation was evident in resting and Ca2+ stimulated Triton X‐100 skinned fibers. Ca2+‐dependent caldesmon phosphorylation was partially sensitive to the ERK MAP kinase inhibitor PD98059, whereas all caldesmon phosphorylation was sensitive to the general kinase inhibitor, staurosporine. Histamine increased caldesmon phosphorylation levels in intact swine carotid artery, which was sensitive to both PD98059 and staurosporine. Histamine increased ERK MAP kinase activity, which was reversed by PD98059, staurosporine, and EGTA. Histamine‐induced contractions were inhibited by staurosporine but not by PD98059. We interpret these results to suggest that although ERK MAP kinases catalyze caldesmon phosphorylation, a second staurosporine sensitive kinase is also important in caldesmon phosphorylation and it is this pathway that may be more important in contractile regulation. J. Cell. Physiol. 198: 461–469, 2004© 2003 Wiley‐Liss, Inc.

Phorbol 12,13-Dibutyrate-Induced, Protein Kinase C-Mediated Contraction of Rabbit Bladder Smooth Muscle

Contraction of bladder smooth muscle is predominantly initiated by M3 muscarinic receptor-mediated activation of the Gq/11-phospholipase C β-protein kinase C (PKC) and the G12/13-RhoGEF-Rho kinase (ROCK) pathways. However, these pathways and their downstream effectors are not well understood in bladder smooth muscle. We used phorbol 12,13-dibutyrate (PDBu), and 1,2-dioctanoyl-sn-glycerol (DOG), activators of PKC, in this investigation. We were interested in dissecting the role(s) of PKC and to clarify the signaling pathways in bladder smooth muscle contraction, especially the potential cross-talk with ROCK and their downstream effectors in regulating myosin light chain phosphatase activity and force. To achieve this goal, the study was performed in the presence or absence of the PKC inhibitor bisindolylmaleimide-1 (Bis) or the ROCK inhibitor H-1152. Phosphorylation levels of Thr38-CPI-17 and Thr696/Thr850 myosin phosphatase target subunit (MYPT1) were measured during PDBu or DOG stimulation using site specific antibodies. PDBu-induced contraction in bladder smooth muscle involved both activation of PKC and PKC-dependent activation of ROCK. CPI-17 as a major downstream effector, is phosphorylated by PKC and ROCK during PDBu and DOG stimulation. Our results suggest that Thr696 and Thr850-MYPT1 phosphorylation are not involved in the regulation of a PDBu-induced contraction. The results also demonstrate that bladder smooth muscle contains a constitutively active isoform of ROCK that may play an important role in the regulation of bladder smooth muscle basal tone. Together with the results from our previous study, we developed a working model to describe the complex signaling pathways that regulate contraction of bladder smooth muscle.

siRNA-mediated knockdown of h-caldesmon in vascular smooth muscle

Smooth muscle contraction involves phosphorylation of the regulatory myosin light chain. However, this thick-filament system of regulation cannot account for all aspects of a smooth muscle contraction. An alternate site of contractile regulation may be in the thin-filament-associated proteins, in particular caldesmon. Caldesmon has been proposed to be an inhibitory protein that acts either as a brake to stop any increase in resting or basal tone, or as a modulatory protein during contraction. The goal of this study was to use short interfering RNA technology to decrease the levels of the smooth muscle-specific isoform of caldesmon in intact vascular smooth muscle tissue to determine more carefully what role(s) caldesmon has in smooth muscle regulation. Intact strips of vascular tissue depleted of caldesmon produced significant levels of shortening velocity, indicative of cross-bridge cycling, in the unstimulated tissue and exhibited lower levels of contractile force to histamine. Our results also suggest that caldesmon does not play a role in the cooperative activation of unphosphorylated cross bridges by phosphorylated cross bridges. The velocity of shortening of the constitutively active tissue and the high basal values of myosin light chain phosphorylation suggest that h-caldesmon in vivo acts as a brake against contractions due to basally phosphorylated myosin. It is also possible that phosphorylation of h-caldesmon alone in the resting state may be a mechanism to produce increases in force without stimulation and increases in calcium. Disinhibition of h-caldesmon by phosphorylation would then allow force to be developed by activated myosin in the resting state.

Effect of Estrogen on Molecular and Functional Characteristics of the Rodent Vaginal Muscularis

INTRODUCTION Vaginal atrophy is a consequence of menopause; however, little is known concerning the effect of a decrease in systemic estrogen on vaginal smooth muscle structure and function. As the incidence of pelvic floor disorders increases with age, it is important to determine if estrogen regulates the molecular composition and contractility of the vaginal muscularis. AIM The goal of this study was to determine the effect of estrogen on molecular and functional characteristics of the vaginal muscularis utilizing a rodent model of surgical menopause. METHODS Three- to 4-month old Sprague-Dawley rats underwent sham laparotomy (Sham, N = 18) or ovariectomy (Ovx, N = 39). Two weeks following surgery, animals received a subcutaneous osmotic pump containing vehicle (Sham, Ovx) or 17β-estradiol (Ovx). Animals were euthanized 1 week later, and the proximal vagina was collected for analysis of contractile protein expression and in vitro studies of contractility. Measurements were analyzed using a one-way analysis of variance followed by Tukeys post hoc analysis (α = 0.05). MAIN OUTCOME MEASURES Protein and mRNA transcript expression levels of contractile proteins, in vitro measurements of vaginal contractility. RESULTS Ovariectomy decreased the expression of carboxyl-terminal myosin heavy chain isoform (SM1) and h-caldesmon and reduced the amplitude of contraction of the vaginal muscularis in response to KCl. Estradiol replacement reversed these changes. No differences were detected in the % vaginal muscularis, mRNA transcript expression of amino-terminal MHC isoforms, l-caldesmon expression, and maximal velocity of shortening. CONCLUSION Systemic estrogen replacement restores functional and molecular characteristics of the vaginal muscularis of ovariectomized rats. Our results indicate that menopause is associated with changes in the vaginal muscularis, which may contribute to the increased incidence of pelvic floor disorders with age.