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W T Gerthoffer

Publications and source records attributed to W T Gerthoffer.

15 recordsLinked to original sources
PubMed1992

Ca2+ sensitivity of contractile activation during muscarinic stimulation of tracheal muscle.

The muscarinic agonists acetylcholine (ACh) and McN-A-343 act on a homogenous population of M3 receptors in canine tracheal smooth muscle; however, ACh is more effective at releasing stored Ca2+ and at stimulating inositol phosphate production. The effects of ACh and McN-A-343 on intracellular Ca2+ concentration ([Ca2+]i), myosin light chain (MLC) phosphorylation, active stress, and isotonic shortening velocity were compared to determine whether differences in their potency at stimulating second messenger pathways affected their ability to modulate the Ca2+ sensitivity of contractile filament activation. There were no differences in [Ca2+]i or isometric stress during the steady-state phase of submaximal contractions induced by ACh and McN-A-343. ACh produced slightly higher levels of MLC phosphorylation than McN-A-343; these levels were associated with much higher rates of isotonic shortening. This could indicate either an extremely high sensitivity of the shortening velocity to differences in MLC phosphorylation or that mechanisms other than MLC phosphorylation contribute to the regulation of shortening velocity. Results show that receptor-coupled pathways can modulate the relationship between [Ca2+]i and isotonic shortening velocity independently of the relationship between [Ca2+]i and isometric stress.

(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethy↗
PubMed1992

An abnormal rate of actin myosin cross-bridge cycling in colonic smooth muscle associated with experimental colitis.

Previous studies showed that colonic smooth muscle develops less contractile force to neurohumoral stimulation when associated with mucosal inflammation. This study evaluated 1) the Ca2+ dependence for colonic smooth muscle contraction, 2) the maximum velocity of muscle shortening (Vmax), and 3) changes in 20-kDa myosin light-chain (MLC) phosphorylation in distal circular colonic muscle from healthy rabbits and from rabbits with experimental colitis, induced by Formalin and immune complexes. The isometric tension of unskinned muscle stimulated with bethanechol or KCl was less (P less than 0.05) in animals with colitis compared with the control group. In saponin-skinned muscle, the amplitude of the maximal tension at [Ca2+] of 3 x 10(-7) M was decreased (P less than 0.05) in colitis animals (4.3 +/- 0.9 x 10(4) N/m2, n = 7) compared with healthy animals (10.5 +/- 2.4 x 10(4) N/m2, n = 6). However, the ED50 for Ca2+ stimulation was similar (P greater than 0.05) in both groups. When MLC was thiophosphorylated with ATP gamma S, the tension development was decreased in colitis (2.1 +/- 0.3 x 10(4) N/m2, n = 5; P less than 0.01) compared with normals (5.0 +/- 1.4 x 10(4) N/m2, n = 5). In healthy animals, phosphorylation of 20-kDa MLC increased rapidly to 51.2 +/- 3.1% within 15 s after stimulation and subsequently declined to 19.0 +/- 2.1% at 5 min. Vmax was maximal (0.14 Lo/s) 13 s after stimulation and declined before maximal active isometric stress. In colitis animals, the 20-kDa MLC phosphorylation (P less than 0.05) and the Vmax (P less than 0.01) were decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1991

Time-dependent changes in Ca2+ sensitivity during phasic contraction of canine antral smooth muscle.

1. Relationships between cytosolic Ca2+ concentration ([Ca2+]cyt), myosin light chain (MLC) phosphorylation and muscle tension were examined in circular smooth muscle of canine gastric antrum. 2. Electrical slow waves induced a transient increase in [Ca2+]cyt and muscle tension. [Ca2+]cyt increased before the initiation of contraction and reached a maximum before the peak of the phasic contractions. Following the first Ca2+ transient, a second rise in [Ca2+]cyt was often observed. The second Ca2+ transient was of similar magnitude to the first, but only in some cases was this increase in [Ca2+]cyt associated with a second phase of contraction. Relaxation occurred more rapidly than the restoration of resting levels of [Ca2+]cyt. 3. Acetylcholine (ACh; 3 x 10(-7) M) increased the amplitude of Ca2+ transients, caused MLC phosphorylation and increased the force of contraction. The decay of contraction and MLC dephosphorylation preceded that of [Ca2+]cyt. 4. Increasing external K+ (to 25-40 mM) caused a sustained increase in [Ca2+]cyt, but little change in resting tension. This suggests that the Ca2+ sensitivity decreased as [Ca2+]cyt increased. Increasing K+ to 59.5 mM further increased the level of [Ca2+]cyt, induced MLC phosphorylation and caused a transient contraction. When normal levels of K+ were restored, the rates of MLC dephosphorylation and relaxation exceeded the rate of decay in [Ca2+]cyt. 5. Removal of external Ca2+ in depolarized muscles decreased [Ca2+]cyt below the resting level without affecting resting tension. Readmission of Ca2+ to depolarized muscles caused force to develop at [Ca2+]cyt levels below the original resting level, suggesting that Ca2+ sensitivity was increased when the resting level of [Ca2+]cyt was decreased. 6. The phosphatase inhibitor, calyculin-A (10(-6) M), induced tonic contraction and MLC phosphorylation without an increase in [Ca2+]cyt. During these contractures, electrical activity caused transient increases in [Ca2+]cyt and phasic contractions which were superimposed upon the Ca(2+)-independent contracture. In the presence of calyculin-A, relaxation occurred in two phases. The initial, rapid phase of relaxation was not significantly affected by calyculin-A, but the slow phase was significantly decreased. 7. These results suggest that the relationship between [Ca2+]cyt, MLC phosphorylation and contraction changes as a function of [Ca2+]cyt in canine antral muscles. This may be due to a Ca(2+)-and time-dependent phosphatase that regulates the level of myosin phosphorylation.

Acetylcholine↗
PubMed1991

Myosin phosphorylation and calcium in tonic and phasic contractions of colonic smooth muscle.

The time dependence of lightly loaded shortening velocity, myosin phosphorylation, and changes in myoplasmic Ca2+ concentration ([Ca2+]i) were measured during tonic and phasic contractions of circular smooth muscle from the proximal colon of the dog. Shortening velocity was measured by quick release to a 10% afterload. Myosin phosphorylation was measured by an immunoblot method, and changes in [Ca2+]i were estimated by measuring fluorescence intensity at 550 nm in muscle strips loaded with fluo-3. During tonic contractions induced by 60 mM K+, phosphorylation increased monotonically from 0.11 +/- 0.011 to 0.29 +/- 0.015 mol Pi/mol light chain at 10 min. In contrast, lightly loaded shortening velocity increased rapidly within 10 s to 0.042 +/- 0.003 lengths/s and decreased exponentially to 0.013 +/- 0.001 lengths/s at 15 min. During transient contractions induced by 100 microM acetylcholine, phosphorylation increased from 0.16 +/- 0.03 to 0.30 +/- 0.06 mol Pi/mol light chain at 19 s. In contrast, shortening velocity increased to 0.068 +/- 0.015 lengths/s within 2.4 s and decreased significantly to 0.027 +/- 0.009 lengths/s at 22 s. Fluo-3 fluorescence increased in parallel with force during both tonic and transient contractions. In a smooth muscle that is able to contract both tonically and phasically we observed transient increases in shortening velocity without concurrent phosphorylation or [Ca2+]i transients. Therefore, there are factors in addition to myosin phosphorylation or changes in [Ca2+]i that regulate cross-bridge cycling rates in both tonic and phasic contractions.

Acetylcholine↗
PubMed1991

Regulation of the contractile element of airway smooth muscle.

Smooth muscle of the mammalian airways controls airway diameter and resistance to airflow. Smooth muscle tone is in turn controlled by a variety of external signals that are transduced to useful work by contractile proteins. The protein components of the contractile element of airway smooth muscle are similar to those found in other smooth muscles and include actin, myosin, tropomyosin, caldesmon, and calponin. There has been significant recent progress in studies of contractile system regulation of airway smooth muscle. Regulation of myosin light chain kinase, identification of the sites phosphorylated on the regulatory myosin light chains, and the effect of myosin phosphorylation on stress development and crossbridge cycling rates have all been studied in some detail. We infer from these studies that besides myosin phosphorylation there is an important role for a thin filament Ca(2+)-dependent regulatory mechanism. The potentially important thin filament proteins caldesmon and calponin are present in tracheal smooth muscle and may be phosphorylated during contraction. The use of intracellular Ca2+ indicators to estimate changes in intracellular Ca2+ ([Ca2+]i) and the development of several skinned fiber preparations have broadened the scope of physiological studies with airway smooth muscle and have suggested that the contractile element sensitivity to Ca2+ is not fixed but might be modulated by undefined messengers or excitation-contraction pathways. This adds an additional challenge to the continuing effort to define the messengers and regulatory proteins that couple activation of membrane receptors to the contractile element in airway smooth muscle.

Actins↗
PubMed1990

Dissociation between myosin phosphorylation and shortening velocity in canine trachea.

The relationship between glycogen phosphorylase activity (an index of cytosolic Ca2+ content), myosin light-chain phosphorylation, isotonic shortening velocity, and isometric tension was examined in canine trachealis. Responses were measured in tracheal strips contracted with various concentrations of methacholine or K+. Both agonists produced prolonged and concentration-dependent increases in isometric tension that reached 90% of the plateau level within 1 (methacholine) to 5 (K+) min and remained stable over 60 min. In contrast to the monotonic increase in isometric tension, shortening velocity reached a maximum almost immediately (12-48 s) after the addition of either methacholine or K+ and then declined over time to a steady-state level that was 25-40% of the peak. Phosphorylase activity also increased transiently, reaching a maximum 1-2 min after the addition of either agonist before declining to near-basal levels over the 60-min observation period. Unlike the increases in shortening velocity and phosphorylase activity, agonist-induced myosin phosphorylation was not markedly transient. Moreover, regardless of the contractile agonist used, no correlation was found between myosin phosphorylation and shortening velocity when these parameters were compared at corresponding time points. This suggests that myosin phosphorylation is not the sole determinant of shortening velocity in canine trachealis.

Animals↗
PubMed1989

Intestinal transport in megacolonic mice. Alterations in sugar absorption.

This study examined jejunal sugar absorption in piebald mice with congenital megacolon and in normal littermates. Transmural potential difference, short-circuit current, and tissue conductance of flat sheets of jejunum set up in flux chambers were significantly greater in the diseased mice compared to normal siblings. In piebald mice, net absorption of 3-o-methylglucose was enhanced due to a significant increase in mucosal-to-serosal flux compared with normal littermates. Stimulation of electrogenic sodium absorption by alanine (10 mM) increased basal short-circuit currents more in piebald tissues than in tissues from normal mice, whereas stimulation of ion transport by carbachol (10 microM) evoked an increase in short-circuit current that was similar in the two groups. Alterations in intestinal mass, morphology, or Na+, K+-ATPase activity could not account for the increase in absorptive function characteristic of piebald mice.

3-O-Methylglucose↗
PubMed1989

Aequorin luminescence, myosin phosphorylation, and active stress in tracheal smooth muscle.

During muscarinic activation of canine tracheal smooth muscle with carbachol, myosin phosphorylation is significantly more sensitive than stress to the external Ca2+ concentration ([Ca2+]o) [W. T. Gerthoffer. Am. J. Physiol. 250 (Cell Physiol. 19): C597-C604, 1986]. To determine whether the intracellular Ca2+ concentration ([Ca2+]i) correlated more closely with changes in phosphorylation or force, we measured isometric force and light emitted by the luminescent intracellular Ca2+ indicator aequorin as [Ca2+]o was increased in the presence of 1 microM carbachol or 60 mM K+. Myosin phosphorylation was measured using an immunoblot assay in a second set of muscle strips treated identically. Stimulation with carbachol increased aequorin luminescence slightly in strips incubated in Ca2+-free solution. Active stress and aequorin luminescence subsequently increased in parallel as [Ca2+]o was increased. Myosin phosphorylation at 0.05 mM [Ca2+]o (0.30 +/- 0.04 mol Pi/mol light chain) was significantly higher than phosphorylation in Ca2+-free solution with no carbachol (0.12 +/- 0.048 mol Pi/mol light chain) and increased to a maximum of 0.56 +/- 0.03 mol Pi/mol light chain at 1.6 mM [Ca2+]o. In contrast, active stress and aequorin luminescence remained low at low [Ca2+]o and reached a maximum at 2.4 mM [Ca2+]o. Stimulation with carbachol produced greater increases in myosin phosphorylation and active stress for a given change in aequorin luminescence than did K+ depolarization. Stimulation with carbachol also produced a different phosphorylation-stress relationship than did K+ depolarization. These observations are consistent with the possibility that carbachol induces increases in the Ca2+ sensitivity of contractile proteins in tracheal smooth muscle.

Aequorin↗
PubMed1989

Mechanical properties of carotid arteries from DOCA hypertensive swine.

Carotid arteries from control and deoxycorticosterone acetate (DOCA) hypertensive swine were examined for alterations in structure and in contractile properties. Vessels were excised 7 weeks after subcutaneous implantation of the steroid and subsequent elevation in mean arterial pressure from 102 to 133 mm Hg. The carotid media was 1.8 times thicker in arteries from hypertensive animals than in arteries from control animals. This enlargement was associated with an increase in muscle mass, as the fraction of the media composed of smooth muscle cells remained unchanged. Maximal active stress induced by several agonists normalized for cell cross-sectional area was unaltered. No change was observed in sensitivity or maximal response to norepinephrine, histamine, or KCl depolarization. Isotonic shortening rates were also comparable, as was the time course of shortening velocity to a constant afterload during tonic contractions. It is concluded that an enlargement of the carotid media develops in this model of hypertension. However, this response is not associated with detectable alterations in contractile system function.

Animals↗
PubMed1988

Inhibition of tracheal smooth muscle contraction and myosin phosphorylation by ryanodine.

Previous studies have shown that muscarinic activation of airway smooth muscle in low Ca++ solutions increases myosin phosphorylation without increasing tension. Blocking Ca++ influx reduced phosphorylation, but not to basal levels. It was proposed that release of intracellular Ca++ contributed to dissociation of phosphorylation and contraction. To test this hypothesis the effects of ryanodine were studied under similar conditions. Ryanodine (10(-7) to 10(-5) M) antagonized caffeine-induced contraction of canine tracheal smooth muscle. Ryanodine also reduced carbachol-induced contractions and carbachol-induced myosin phosphorylation. The effect of ryanodine on potassium and serotonin-induced contractions was also investigated to test for a nonspecific inhibitory effect. In contrast to the effect on carbachol responses, ryanodine (10(-5) M) potentiated the contractile response to low concentrations of serotonin and potassium, but had no effect on the maximum response to either stimulant. Carbachol (10(-6) M) and ryanodine (10(-5) M) both significantly decreased 45Ca++ content of tracheal muscle. The effect of ryanodine and carbachol together on 45Ca++ content was not greater than either drug alone suggesting that ryanodine reduces the caffeine and carbachol responses by depleting releaseable Ca++ stores. Ryanodine significantly reduced Ca++-induced contraction and myosin phosphorylation in carbachol-stimulated muscle, suggesting that some of the Ca++ responsible for elevated phosphorylation is released from the sarcoplasmic reticulum.

Alkaloids↗
PubMed1987

Effects of potassium and norepinephrine on calcium influx in guinea-pig vas deferens.

The effects of potassium (K+) and adrenoceptor agonists on 45Ca influx and contraction of isolated vas deferens from reserpine-treated guinea-pigs has been investigated. K+ (18-84 mM) increased 45Ca influx with the maximum influx occurring at 50 mM K+. The rate of influx upon exposure to K+ was highest in the first 2 min and decreased thereafter. In contrast to K+, norepinephrine, methoxamine and xylazine did not increase the rate of 45Ca influx. Norepinephrine and methoxamine, but not xylazine, inhibited 45Ca influx induced by high K+. The inhibitory effect of norepinephrine was also present in denervated tissues. Prazosin, but not yohimbine or propranolol, blocked the inhibitory effect of norepinephrine. Potassium-induced contractions were significantly potentiated in the presence of norepinephrine, especially at low calcium concentrations. We conclude that K+ contracts the guinea-pig vas deferens by increasing the influx of calcium whereas stimulation of alpha 1-adrenoceptors prevents 45Ca influx elicited by high potassium.

Adrenergic alpha-Agonists↗
PubMed1987

Selectivity of phenytoin and dihydropyridine calcium channel blockers for relaxation of the basilar artery.

We addressed the questions of whether or not phenytoin is a direct vasodilator and if it is selective for brain blood vessels, by studying the relaxant effects of phenytoin on isolated segments of canine basilar, femoral, and brachial arteries. Two dihydropyridine calcium channel blockers, nifedipine and PY 108-068, were also studied for comparison with phenytoin and to test for cerebral selectivity. Blood vessels were contracted with K+, prostaglandin F2 alpha, or serotonin. Phenytoin relaxed the basilar artery with low potency (pD2, 4.71 +/- 0.14) and moderate selectivity. Phenytoin also antagonized Bay K 8644 contractions of basilar artery in a noncompetitive manner. Basilar arteries contracted with 60 mM K+ were the most sensitive to nifedipine (pD2, 8.72 +/- 0.18), followed by the mesenteric (pD2, 8.24 +/- 0.07), femoral (pD2, 8.04 +/- 0.18), and brachial (pD2, 7.66 +/- 0.23) arteries. A similar pattern was observed in potassium-depolarized arteries relaxed by PY 108-068. The calcium dependence of contraction was studied using intact muscles depolarized in 60 mM K+ as well as chemically skinned basilar artery. Mean pD2 values for Ca2+-induced contractions of intact, depolarized arteries were not different (basilar, 4.15 +/- 0.13; mesenteric, 4.04 +/- 0.07; femoral, 4.24 +/- 0.11). The mean Ca2+ EC50 of chemically skinned basilar arteries was 8.7 X 10(-7) M, which is similar to the Ca2+ sensitivity of other skinned smooth muscles. The beneficial effect of phenytoin in treating cerebral ischemia may be due in part to relaxation of vascular smooth muscle. The dihydropyridines were potent smooth muscle relaxants with selectivity for the basilar artery.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1987

Dissociation of myosin phosphorylation and active tension during muscarinic stimulation of tracheal smooth muscle.

The Ca dependence of contraction and myosin phosphorylation was investigated in canine tracheal smooth muscle stimulated with carbachol, K or serotonin. Previous studies of tracheal muscle showed carbachol concentration-response curves for contraction and myosin phosphorylation were superposable. In contrast, there was a striking difference in the Ca++ sensitivities of tension and myosin phosphorylation when Ca++ concentration-response curves were constructed in the presence of 10(-7) M carbachol. Significant phosphorylation (greater than 0.3 moles phosphate/mole 20,000 dalton myosin light chain) was observed in the absence of active tension. In the present study, carbachol (10(-7) and 10(-6) M) and serotonin (10(-5) M) also induced significant myosin phosphorylation in low Ca++ solutions (0-0.025 mM CaCl2) without proportional increases in tension. K+ depolarization in Ca++-free physiological salt solution (60 mM KCl, 10(-6) M atropine) yielded phosphorylation not significantly different from basal levels. All stimulants induced active stress after readmission of Ca. The Ca++ dependence curve for myosin phosphorylation in muscles stimulated with carbachol was shifted up and to the left of the force curve. Atropine (10(-6) M) significantly reduced phosphorylation induced by carbachol in Ca++-free solutions, as did 3 X 10(-6) M nifedipine and 10 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Phorbol 12-myristate, 13-acetate or phorbol 12,13-dibutyrate did not increase basal phosphorylation or phosphorylation in low Ca++ solutions, suggesting that protein kinase C did not phosphorylate myosin in this case. Myosin phosphorylation under these conditions is not sufficient to support contraction, and is reduced by treatments that decrease Ca++ entry.(ABSTRACT TRUNCATED AT 250 WORDS)

Atropine↗