Ralph C. Kolbeck

Apparent irrelevance of cyclic nucleotides to the relaxation of tracheal smooth muscle induced by theophylline

The mechanism of action of the negative inotropic (relaxing) effect of the methylxanthine theophylline on respiratory smooth muscle is generally assumed to involve inhibition of cyclic nucleotide phosphodiesterase. When guinea pig tracheal rings or dog tracheal muscle strips were incubated with low concentrations of theophylline capable of producing a significant negative inotropic effect in vivo and in vitro, there was no alteration of cyclic 3′,5′-adenosine monophosphate (cAMP) or cyclic 3′,5′-guanosine monophosphate (cGMP) levels.45Ca uptake increased however, and there was a subcellular redistribution of elemental calcium consistent with a sequestration of calcium in mitochondria and a decrease of myoplasmic calcium ion (Ca++) concentration.

Influence of ageing on the fatigability of isolated mouse skeletal muscles from mature and aged mice

We investigated the influence of ageing on the fatiguing characteristics of the mouse extensor digitorum longus (EDL) muscle as compared to those of the soleus muscle. Fatigue was produced by an intermittent stimulation protocol. We report for mature and aged animals the effects of fatigue on force produced during stimulation patterns that in non‐fatigued muscle gave maximum force (Tmax, high frequency stimulation) and approximately half‐maximum force (1/2Tmax, low frequency stimulation). In 15‐month‐old (mature) mice, fatiguing stimulation decreased Tmax in EDL and soleus muscle to 10.3 ± 1.0% and 33.4 ± 3.0% of control, respectively. In 30‐month‐old (aged) mice, the decrease in Tmax in EDL and soleus was statistically equal to that of the younger animals. Fatiguing stimulation decreased 1/2Tmax in EDL and soleus from 15‐month‐old animals to 22.5 ± 2.9% and 45.7 ± 0.3% of control, respectively. In 30‐month‐old animals, the 1/2Tmax in EDL and soleus muscle decreased to 18.2 ± 1.3% and 35.0 ± 3.6% of control, respectively. Under all conditions, the soleus fatigued significantly less. Contractile recovery from fatiguing stimulation was complete for the soleus in both age groups after 30 min, but incomplete for the EDL. The 1/2Tmax/Tmax ratio significantly increased in EDL and soleus muscle from 15‐month‐old animals after fatiguing stimulation. This increase was less significant in EDL, and absent in soleus muscle, from 30‐month‐old animals. These results indicate that fatiguing stimulation induces a leftward shift in the force‐frequency relationship in the young animals; this shift is either significantly less (EDL) or absent (soleus) in the older animals. We speculate that the leftward shift of the force‐frequency relationship may reflect a protective mechanism in younger animals against some of the damaging effects of fatiguing stimulation (i.e. oxidative stress).

Studies of mediator involvement in cooling-induced alterations of guinea pig tracheal smooth muscle contractility.

Abstract Heat loss from airway smooth muscle is a potent stimulus which causes substantial, but poorly understood, alterations in muscle tension. This study considered the involvement of endogenous mediators in cooling-induced tension changes in incubated guinea pig trachea. Smooth muscle tension was monitored in tracheal cylinders which were carefully cooled from 37 to 30°C in the presence or absence of various inotropic mediators. In our study, cooling alone, at a rate of 1 °C/min, was associated with an average loss of smooth muscle tension of 88.2 mg. Cooling tracheal tissue that had been previously exposed to 3 × 10-6 M histamine, however, caused an additional increase in tracheal tension of 133 mg, over and above that caused by histamine alone. In the presence of 10-5 M prostaglandin F2α, or 10-5 M thromboxane B2, cooling was associated with respective losses of smooth muscle tension of 211.4 and 211.2 mg, as compared to the tension associated with these mediators when they were used alone under control conditions. When the speed of tracheal cooling was increased to 40°C/min, there was a slight increase in tension for 20 sec followed by a pronounced and sustained relaxation. The mechanisms involved in the response of airway smooth muscle to cooling are complex. The results of our study, however, suggest that mediators may play a role in the cooling-induced alterations of airway smooth muscle tension.

Alkalotic disequilibrium in patients with solid tumors: Rediscovery of an old finding

Abstract Acid-base equilibrium in two groups of cancer patients, one with newly diagnosed bronchogenic cancer and one with various solid tumors, was compared to that of a group of normal volunteers and a group of hospitalized patients with diverse diseases. A consistent tendency toward alkalosis was found in both groups of cancer patients. Similar findings reported in the literature of 40–70 years ago are discussed and the possible biochemical implications of these findings considered. It is our hope that this report of findings obtained with modern technology will encourage studies of the effects of alkalinity in the pathogenesis and progression of cancer.

Crown ethers which influence cardiac and respiratory muscle contractility

Guinea-pig tracheal smooth muscle and heart muscle demonstrated a variety of in vitro positive and negative inotropic responses to concentrations of crown ethers in the nmole/l to μmole/l range. It is suggested that these ionophoretic compounds have potential as therapeutic agents.

Inotropic influence of macrocyclic polyethers on tracheal smooth muscle

Incubated guinea pig tracheal smooth muscle exhibited both positive and negative inotropic responses to a variety of crown ether analogs that ranged in size from 12-crown-4 to 30-crown-10 and included molecules whose lipophilicity was modified by the addition of benzo- and cyclohexo-substituents on the basic molecular framework. The inotropic influence of crown ethers may not only be due to their ionophoretic capabilities but may result from their ability to affect alterations in membrane physiology.

Nonpolar density gradient ultracentrifugation in the direct determination of myocardial subcellular calcium.

A new method is described for making direct measurements of compartmentalized subcellular stores of calcium in the isolated perfused dog heart. Cellular calcium was immobilized by freezing the myocardium in diastole with an extremely cold fluorocarbon fluid (-125 degrees C). All subsequent procedures were conducted under conditions which prevented ionic diffusion, either at temperatures well below the freezing point of water or in the absence of water. Sarcolemmal and mitochondrial enriched fractions were segregated from dessicated, homogenized, myocardial tissue by ultracentrifugation utilizing density-gradients composed of blends of silicone and halocarbon on fluids within which physiological salts are insoluble. The total calcium content of these isolated fractions were then determined by atomic absorption spectrophotometry. Ouabain and epinephrine were subsequently used to alter the contractility of the perfused hearts and such contractile alterations were then related to changes noted in the calcium activity of the isolated subcellular fractions. In this study the calcium levels of the enriched mitochondrial fractions were elevated by both ouabain and epinephrine, while the calcium levels of the enriched sarcolemmal fractions were elevated only by ouabain. The advantage of this segregative procedure is that it prevents artifactual intercompartmental calcium rearrangement and preserves calcium levels to those initially fixed in situ at the time of freezing.

The influence of lanthanum on the subcellular distribution of calcium in the perfused dog heart

A newly developed method was used to make a direct study of the influence of lanthanum on the subcellular distribution of isotopic and elemental calcium in the isolated perfused dog heart. The trivalent cation was introduced into the heart for the last 5 min of a 60 min perfusion period. Developed pressure and dP/dt were monitored continually from a fluid filled balloon positioned within the left ventricular chamber. At the end of the perfusion period, calcium was fixed within the myocardium by rapid freezing followed by vacuum desiccation at -60 degrees C. Enriched populations of sarcolemma and mitochondria were then obtained utilizing a newly developed nonpolar density gradient ultracentrifugation technique. Lanthanum was found to decrease dP/dt by 82.5% without significant changes in contractile rate. Atomic absorption spectrophotometric analysis revealed that lanthanum was associated with a 48.8% decrease in sarcolemmal calcium and a 159.6% increase in mitochondrial calcium. Lanthanum caused a 40.2% increase in the mitochondrial tissue/medium 45Ca ratio without significantly altering the isotropic activity of the sarcolemma. The results confirm that lanthanum-induced negative inotropy is associated with a displacement of sarcolemma-associated calcium. Surprisingly, the diminished contractility occurs with an increase in mitochondrial-associated calcium.

Temporal relationship of cyclic nucleotide levels and calcium exchange to histamine-induced tension development.

Abstract The purpose of these experiments was to study the temporal relationship between tension development in incubated guinea pig tracheal smooth muscle and changes in tissue levels of cAMP and cGMP, and isotopic Ca. Dose–response studies were performed with increasing concentrations of histamine both in the absence and presence of H1 receptor blockade using 10-5 M diphenhydramine. The time course of tension development was subsequently determined in the presence of three concentrations of histamine shown to cause 50% (3 × 10-6 M), 85% (9 × 10-6 M), and 100% (5 × 10-5 M) of maximal contraction. Tissue cyclic nucleotide and 45Ca levels were measured 20 sec, 1 min, and 6 min after the onset of contraction. For comparison, the influence of carbachol was also studied. Our findings demonstrate that there were no detectable alterations in tissue cAMP or cGMP levels during the initial phases of contractile change. In contrast, tissue isotopic Ca uptake increased early in histamine-induced contraction and was blocked by the H1 antagonist.