Inhibition of mouse neuromuscular transmission and contractile function by okadaic acid and cantharidin. (9/89)

1. Phosphorylations of cellular proteins modulate biological activities. The effects of okadaic acid (0.1 - 10 microM) and cantharidin (1 - 100 microM), inhibitors of protein phosphatases, on the synaptic transmission at the mouse neuromuscular junction were explored. 2. Both inhibitors almost completely depressed twitch forces elicited by electrical stimulation of diaphragm muscles (the IC(50)s for okadaic acid and cantharidin were 1.1+/-0.2 and 13+/-1 microM, n=5, respectively) and suppressed contractures evoked by high K(+) and ryanodine more than 70%. Contractures caused by cardiotoxin, which destroys the integrity of sarcolemma, were not depressed. 3. Both okadaic acid (10 microM) and cantharidin (100 microM) depolarized muscle membranes from approximately -80 to approximately -60 mV in a partially reversible and tetrodotoxin-sensitive manner. The initial short-term enhancement of twitch responses (up to approximately 40%) was correlated with the inhibitors-induced repetitive firings of muscle action potential. 4. Treatment with either agent resulted in nearly complete inhibitions of endplate potential (epp). The IC(50)s were 0.8+/-0.2 and 9+/-2 microM (n=5), respectively, for okadaic acid and cantharidin. On high frequency stimulation, the coefficient of epps was increased more than 10 fold and the extent of epp run-down during stimulations intensified from approximately 25 to approximately 75%. Analyses of presynaptic quantal releases revealed decreases in epp quantal content and the immediately available vesicle pool. 5. The frequency of miniature epp was initially elevated up to 2 fold then suppressed down to approximately 30%. The small reduction in the amplitude was antagonized when the membrane of endplate area was repolarized. 6. The data suggest that okadaic acid and cantharidin inhibit mobilizations of synaptic vesicles and depress Ca(2+) release from sarcoplasmic reticulum and that protein phosphatases participate in the modulation of motor function.  (+info)

Role of protein phosphatases in regulation of cardiac inotropy and relaxation. (10/89)

We studied the effects of the protein phosphatase (PP) inhibitor cantharidin (Cant) on time parameters and force of contraction (FOC) in isometrically contracting electrically driven guinea pig papillary muscles. We correlated the mechanical parameters of contractility with phosphorylation of the inhibitory subunit of troponin (TnI-P) and with the site-specific phosphorylation of phospholamban (PLB) at serine-16 (PLB-Ser-16) and threonine-17 (PLB-Thr-17). Cant (after 30 min) started to increase FOC (112 +/- 4% of control, n = 10) and TnI-P and PLB-Thr-17 (120 +/- 5 and 128 +/- 7% of control) without any alteration of relaxation time. Cant (10 microM) started to increase PLB-Ser-16, but the relaxation was shortened at only 100 microM (from 140 +/- 9 to 116 +/- 12 ms, n = 9). Moreover, 100 microM Cant, 3 min after application, started to increase PLB-Thr-17, TnI-P, and FOC. Cant (100 microM) began to increase PLB-Ser-16 after 20 min. This was accompanied by shortening of relaxation time. Differences in protein kinase activation or different substrate specificities of PP may explain the difference in Cant-induced site-specific phosphorylation of PLB in isometrically contracting papillary muscles. Moreover, PLB-Thr-17 may be important for inotropy, whereas PLB-Ser-16 could be a major determinant of relaxation time.  (+info)

Genetic and chemical reductions in protein phosphatase activity alter auxin transport, gravity response, and lateral root growth. (11/89)

Auxin transport is required for important growth and developmental processes in plants, including gravity response and lateral root growth. Several lines of evidence suggest that reversible protein phosphorylation regulates auxin transport. Arabidopsis rcn1 mutant seedlings exhibit reduced protein phosphatase 2A activity and defects in differential cell elongation. Here we report that reduced phosphatase activity alters auxin transport and dependent physiological processes in the seedling root. Root basipetal transport was increased in rcn1 or phosphatase inhibitor-treated seedlings but showed normal sensitivity to the auxin transport inhibitor naphthylphthalamic acid (NPA). Phosphatase inhibition reduced root gravity response and delayed the establishment of differential auxin-induced gene expression across a gravity-stimulated root tip. An NPA treatment that reduced basipetal transport in rcn1 and cantharidin-treated wild-type plants also restored a normal gravity response and asymmetric auxin-induced gene expression, indicating that increased basipetal auxin transport impedes gravitropism. Increased auxin transport in rcn1 or phosphatase inhibitor-treated seedlings did not require the AGR1/EIR1/PIN2/WAV6 or AUX1 gene products. In contrast to basipetal transport, root acropetal transport was normal in phosphatase-inhibited seedlings in the absence of NPA, although it showed reduced NPA sensitivity. Lateral root growth also exhibited reduced NPA sensitivity in rcn1 seedlings, consistent with acropetal transport controlling lateral root growth. These results support the role of protein phosphorylation in regulating auxin transport and suggest that the acropetal and basipetal auxin transport streams are differentially regulated.  (+info)

PP1 inhibitors depolarize Hermissenda photoreceptors and reduce K+ currents. (12/89)

Previous research indicates that activation of protein kinase C (PKC) plays a critical role in the induction and maintenance of memory-related changes in neural excitability of Type B photoreceptors in the eyes of nudibranch mollusk Hermissenda crassicornis (H.c.). The enhanced excitability of B cells is due in part to PKC-mediated reduction in somatic K+ currents. Here we examined the effects of protein phosphatase inhibitors on Type B photoreceptor excitability and K+ currents to determine the role(s) of protein phosphatases on memory formation in Hermissenda. Using electrophysiological and pharmacological methods, we found that the PP1 inhibitors calyculin A and inhibitor-2 depolarized Type B photoreceptors by 20-30 mV. A broad-spectrum kinase inhibitor, H7, blocked this effect. The depolarization induced by PP1 inhibition occluded that produced by an in vitro associative conditioning procedure. Calyculin and inhibitor-2 reduced the same B cell K+ currents (I(A) and I(delayed)) that are reduced by in vitro and behavioral conditioning. H7 blocked the reductions. Cantharidic acid (PP2A inhibitor) and cyclosporin (PP2B inhibitor) had negligible effects on B cell resting membrane potential, K+ currents, and in vitro conditioning-produced cumulative depolarization of B cells. These results suggest that the functional activity of K+ channels in B cells is sustained by basal activity of PP1. Inhibiting PP1 appears to allow one or more constitutively active kinase(s) to reduce K+ channel activity and thus mimic the effects of conditioning. Our results suggest that PP1 may oppose and/or constrain the extent of learning-produced changes in B cell excitability.  (+info)

Activation and inactivation of cAMP-response element-mediated gene transcription in cardiac myocytes. (13/89)

OBJECTIVE: Chronic beta-adrenergic stimulation of the cAMP-dependent signalling pathway is implicated in functionally relevant expressional changes in congestive heart failure. We studied activation and inactivation of the cardiac gene transcription mediated by the cAMP-response element (CRE) and the CRE-binding protein (CREB) as an important mechanism of a cAMP-dependent gene regulation. METHODS: We investigated the transcriptional activation by forskolin, an activator of the adenylyl cyclase, in chick embryonic cardiomyocytes transfected with a CRE-controlled luciferase construct in comparison to the phosphorylation and expression of CREB determined on immunoblots. RESULTS: Forskolin (10 micromol/l; 8 h) increased CRE-mediated transcription and phosphorylation of CREB 13- and 1.5-fold, respectively. The phosphorylation was further elevated in combination with cantharidin, an inhibitor of type 1+2A protein phosphatases. The transcriptional response to forskolin was desensitized by pretreatment with forskolin (1 micromol/l; 24 h) while CREB phosphorylation was increased. In forskolin-pretreated cells, total CREB protein levels were decreased. Cantharidin did not restore the attenuated transcriptional response. CONCLUSIONS: In cardiomyocytes, there is an activation of the CRE-mediated gene transcription by forskolin that is attenuated after prolonged stimulation, and this attenuation is not dependent from a dephosphorylation of CREB. We suggest that attenuation of the CRE-mediated transcription through chronic stimulation of the cAMP-pathway, e.g. by elevated catecholamines, contributes to the altered expressional regulation in congestive heart failure.  (+info)

Glucose and cAMP regulate the L-type pyruvate kinase gene by phosphorylation/dephosphorylation of the carbohydrate response element binding protein. (14/89)

Recently we purified and identified a previously uncharacterized transcription factor from rat liver binding to the carbohydrate responsive element of the L-type pyruvate kinase (L-PK) gene. This factor was named carbohydrate responsive element binding protein (ChREBP). ChREBP, essential for L-PK gene transcription, is activated by high glucose and inhibited by cAMP. Here, we demonstrated that (i) nuclear localization signal and basic helix-loop-helix/leucine-zipper domains of ChREBP were essential for the transcription, and (ii) these domains were the targets of regulation by cAMP and glucose. Among three cAMP-dependent protein kinase phosphorylation sites, Ser(196) and Thr(666) were the target sites. Phosphorylation of the former resulted in inactivation of nuclear import, and that of the latter resulted in loss of the DNA-binding activity and L-PK transcription. On the other hand, glucose activated the nuclear import by dephosphorylation of Ser(196) in the cytoplasm and also stimulated the DNA-binding activity by dephosphorylation of Thr(666) in the nucleus. These results thus reveal mechanisms for regulation of ChREBP and the L-PK transcription by excess carbohydrate and cAMP.  (+info)

Pharmacokinetics and inflammatory fluid penetration of intravenous daptomycin in volunteers. (15/89)

The lipopeptide antimicrobial daptomycin was administered intravenously at a dose of 4 mg/kg of body weight to seven healthy male volunteers. The concentrations of daptomycin in plasma, cantharidin-induced inflammatory fluid, and urine were measured by a microbiological assay. The mean +/- standard deviation peak concentrations in plasma and inflammatory fluid were 77.5 +/- 8.3 and 27.6 +/- 9.5 microg/ml, respectively; the mean terminal elimination half-lives were 7.74 and 13.2 h, respectively. The overall penetration of total drug into the inflammatory fluid (measured by ratio of the area under the concentration-time curve from 0 to 24 h for inflammatory fluid compared with that for plasma) was 68.4%. The mean urinary recovery over 24 h was 59.7%.  (+info)

Single-dose pharmacokinetics and penetration of BMS 284756 into an inflammatory exudate. (16/89)

The pharmacokinetics of a single dose of BMS 284756 were determined following oral administration of a 600-mg dose to eight healthy male volunteers. Concentrations of the drug were measured in plasma and a cantharidine-induced inflammatory exudate by a microbiological assay. The mean peak concentration in plasma of 10.4 microg/ml (standard deviation [SD], 1.3 microg/ml) was attained at a mean time of 1.2 h (SD, 0.5 h) after the dose. The penetration into the inflammatory exudate was 82% (SD, 15.7%). A mean peak concentration of 7.2 microg/ml (SD, 2.4 microg/ml) was attained in the inflammatory exudate at 5.3 h (SD, 1.5 h). The elimination half-lives from plasma and inflammatory fluid were 9.8 h (SD, 1.1 h) and 8.5 h (SD, 1.9 h), respectively. The areas under the concentration-time curves for plasma and inflammatory fluid were 96.7 microg x h/ml (SD, 10.3 microg x h/ml) and 77.9 microg x h/ml (SD, 19.2 microg x h/ml), respectively.  (+info)