C. Paul Bianchi

Trace metal (Cu and Zn) adaptation of organ systems of the american eel, Anguilla rostrata, to external concentrations of cadmium

1. The impact of external cadmium on the concentrations of cadmium (Cd), copper (Cu) and zinc (Zn) in seven tissues of the American eel, Anguilla rostrata was investigated. Even after a week in freshwater with undetectable levels of Cd, the tissues of eels caught in fresh and/or brackish waters of the United States east coast contained Cd in kidney, liver, gut, and brain. 2. When the eels were exposed up to 16 weeks to low and high sublethal concentrations of Cd (75 and 150 micrograms/l, respectively), the highest tissue concentrations of Cd were found after two weeks of exposure. The accumulation was dose-related in all tissues studied except for the kidney. After 8 weeks of Cd exposure, the tissue levels of Cd were markedly reduced, and they were in a similarly low range after 16 weeks. At this time, the highest Cd concentrations were found in the gills and kidney. 3. After two weeks of Cd exposure, there was a drop of the tissue concentrations of Cu in liver and heart, and of Zn in gut and liver. The low concentrations of the two metals in other organs did not allow an evaluation of the Cd impact. After 16 weeks, the Cu concentrations in all tissues, with the exception of the liver, were similar to, or even higher than control levels. At the same time, Zn concentrations exceeded the control levels in heart and kidney of eels exposed to 75 and 150 micrograms Cd/l, respectively. 4. It is clear that some tissues of the eel are able to maintain or restore normal levels of Cu and Zn, up to 16 weeks, despite concomitant Cd accumulation.

Further evidence for a neuromodulatory role of FMRFamide involving intracellular Ca2+ pools in smooth muscle of Mytilus edulis

Abstract 1. The present study examined the interaction between FMRFamide (Phe-Met-Arg-Phe-NH 2 ) and neurotransmitter function. 2. An isolated tissue preparation of molluscan smooth muscle (ABRM) from Mytilus edulis was prepared according to standard procedures and the isometric tension produced by acetylcholine (10 −6 –10 −3 M) was measured. 3. FMRFamide (10 −6 M) enhanced the contractions produced by doses of acetylcholine (ACh) below the ed 50 , but had no effect on E max or on contractions produced by ACh at doses greater than the ed 50 . A similar action of FMRFamide was observed on contractions produced by carbachol. 4. FMRFamide responses were less susceptible than ACh responses to inhibition by heavy metal ions. 5. These results are consistent with the concept that FMRFamide may play a modulatory role in neurotransmitter function and suggest that this action might involve the mobilization of a Ca 2+ pool normally inaccessible to ACh.

The effect of diazepam on tension and electrolyte distribution in frog muscle

The effect of diazepam on sartorius muscles of the frog was evaluated. Resting tension in sartorius muscle was not affected by diazepam (5 x 10(-5)M) but twitch tension was increased and tetanus tension decreased. The kinetics of 45Ca efflux were altered by diazepam. The calcium content of the intermediate pool was increased by diazepam (5 x 10(-6)M). When the diazepam concentration was increased (5 x 10(-5)M), the time constant of the slow pool decreased and the 45Ca content of the intermediate pool increased further. It is suggested that diazepam interferes with the calcium sequestering system of the sarcoplasmic reticulum (slow pool) and causes an increase of the calcium content of the myofibrillar space (intermediate pool).

Effect of Pharmacological Agents on Calcium Stores in Amphibian Fast and Slow Muscle Fibers

Amphibian fast and slow muscles muscle fibers differ in regard to structure, innervation, and contractile response. These differences can be utilized to study drug effects on cellular Ca2+ metabolism. The amphibian slow muscle fiber contains multiple end plates, is innervated by small nerve fibers, and gives a graded tonic response to repetitive neural stimulation. Tension is regulated by graded levels of depolarization of the surface membrane (Kuffler and Williams, 1953a, 1953b). Identical contractures produced by K+ depolarization or acetylcholine are associated with a sustained increase in Ca2+ influx, a transient increase in Ca2+ efflux and a net gain of Ca2+ which amounts to 0.24 µmol/g for the KC1 contracture and 0.27 µmol/g for the acetylcholine contracture (Bianchi, 1968a). During relaxation the Ca2+ becomes sequestered within the sarcoplasmic reticulum of the slow muscle fibers. The sarcoplasmic reticulum of the amphibian slow muscle fiber lacks the triad structure of the amphibian fast muscle fiber, but does contain invaginations of the cell membrane which extend into the cell interior and run in a longitudinal fashion parallel to the fiber axis (Page, 1965). The primary function of the sarcoplasmic reticulum present in the amphibian slow muscle fiber is to allow for rapid relaxation following the shutting off of Ca2+ influx during repolarization of the surface membrane. In the relaxed state Ca2+ efflux must exceed influx in order to restore the fiber Ca2+ content to steady state conditions.

Calcium dependent magnesium uptake in myocardium

The frog myocardium maintains magnesium content at a steady state level when stimulated at 0.4Hz while being perfused with Ringers solution containing 1 x 10(-3) M Ca2+ and 5 x 10(-7) M magnesium. When calcium is removed 43% of tissue magnesium is lost within 30 seconds or 12 beats. Restoration of calcium to the perfusion solution causes reaccumulation of magnesium from a solution containing 5 x 10(-7) M magnesium. The reaccumulation of magnesium indicates a highly selective transport system for magnesium which is dependent upon the presence of calcium. Calcium appears to reduce the leak of magnesium from the myocardium and enhances the transport of magnesium into the myocardial cell. Intracellular magnesium is a necessary cofactor for hundreds of enzymes, and is essential for protein synthesis and as an extracellular divalent cation helps to stabilize excitable membranes in conjunction with calcium. The concentration of ionized magnesium in the sarcoplasm of myocardial muscle has an average value of 1.45 mM +/- 1.37 (standard deviation), N = 19) with a range of 0.5 to 3.6 mM (1). The heart with its numerous mitochondria and high enzymatic activity is vulnerable to myocardial damage due to magnesium loss. The isolated frog ventricle conserves intracellular magnesium when perfused with Ringers solution containing no added magnesium and maintains function for hours. The ability to conserve magnesium suggests a low permeability of the sarcolemma to magnesium and an extremely efficient inward transport system. Removal of calcium as well as magnesium from the perfusion solution causes a rapid loss of tension in the electrically driven frog ventricle (0.4) Hz.(ABSTRACT TRUNCATED AT 250 WORDS)

Bimodal operation of the ryanodine-sensitive transducer calcium channel

Ryanodine binds to the transducer calcium channel complex that links depolarization of the transverse tubule to calcium release from the terminal cisternae during excitation-contraction coupling of skeletal muscle. Ryanodine exerts a bimodal action on the transducer calcium channel complex depending upon membrane potential and concentration. When the transmembrane potential is at resting level (-90 mV inside cell vs outside), low concentrations of ryanodine 10(-10) M to 10(-8) M favor calcium influx from outside which in turn causes calcium release from the terminal cisternae via calcium operated calcium channels. The leak from the terminal cisternae is insufficient to cause contraction but does cause a large increase in aerobic energy utilization by the Ca-ATPase of the sarcoplasmic reticulum. When the transmembrane potential is made more positive (-40 mV) the transducer channel is opened to the terminal cisternae of sarcoplasmic reticulum and is maintained in an open state by ryanodine allowing calcium efflux from the terminal cisternae to the sarcoplasm. At higher concentrations of ryanodine the transducer-calcium channel becomes open to the terminal cisternae and its store of ionized calcium leaks from the terminal cisternae in sufficient quantities to cause a contracture. The ryanodine-sensitive calcium transducer calcium channel operates in a bimodal manner. At low concentrations less than 10(-4) M the ryanodine-sensitive transducer calcium channel is open to the lumen of the T-tubule and allows calcium to flow in and trigger further calcium release. At higher concentrations the ryanodine-sensitive transducer channel opens to allow a calcium efflux from the terminal cisternae in sufficient quantities to cause contracture.

Conformation state of the ryanodine receptor and functional effects of ryanodine on skeletal muscle

The ryanodine receptor (RyR) and the dihydropyridine (DHP) receptor (L-channels) comprise the main elements of the functional feet of the triadic element in skeletal muscle. These two main elements have conformational states that are regulated by the membrane potential and the consequent electrical field. The pharmacological action of ryanodine on skeletal muscle depends upon the physiological functional state of the RyR. At a resting potential of -90 m V, ryanodine at very low concentrations, 10(-11) M, causes the RyR to have a low conductance state which allows calcium to leak from the terminal cisternae of the sarcoplasmic reticulum and to be recycled with ATP utilization, leading to a marked increase in oxygen consumption and aerobic metabolism. At concentrations greater than 10(-6) M, ryanodine can cause a slowly developing contracture of resting muscle, inhibit the muscle twitch when the RyR complex is formed during stimulation, and, if formed before stimulation, accelerate the development of contracture. Biochemical studies have revealed that the RyR has four binding sites in which the conductance state depends upon the number of sites occupied by ryanodine. Our present understanding of the RyR-operated calcium channel is the result of an interdisciplinary approach in which each discipline (anatomy, physiology, biophysics, and biochemistry) contributes to our knowledge of the pharmacological action of ryanodine.

Cisplatinum enhancement of myocardial Mg2+ transport

Cisplatinum in a concentration (4.3 x 10(-6) M) corresponding to the therapeutic plasma concentration for cancer patients was found to cause a marked enhancement of magnesium efflux and uptake in perfused frog myocardium. The magnesium content of the perfused frog ventricle is increased from 6.66 +/- 0.34 mumol/g wet wgt to 8.03 +/- 0.38 mu mol/g wet wgt. Cisplatinum had a negative inotropic action reducing contractile force to 46 +/- 8% of initial force after 40 min of perfusion. The corresponding control contractile force was reduced to 74 +/- 7%. Removal of calcium and magnesium from the perfusion solution containing 0.5 mM EDTA for 10 minutes caused contractile force to be reduced to 0 after 6 beats at 24 min-1. After ten minutes of perfusion with EDTA, 1.87 mu mol/g wet wgt of magnesium was lost from control ventricles. Cisplatinum increased the loss to 4.08 +/- 0.34 mu mol/g wet wgt. The magnesium lost during EDTA perfusion was completely recovered after 5 minutes of perfusion in Ringer or Tyrode solution by both control and cisplatinum treated frog ventricles. The contractile force also recovered to the level prior to perfusion with EDTA Ringer. The rate of Mg2+ efflux in EDTA Ringer is largest during the first 3 minutes and was 0.170 +/- 0.051 p mol cm-2 sec-1 for controls and 0.798 p mol cm-2 sec-1 for the cisplatinum treated ventricles. During the last 7 min of perfusion in EDTA Ringer the Mg2+ efflux was reduced to 0.057 +/- 0.005 p mol cm-2 sec-1 for control ventricles and 0.170 p mol cm-2 sec-1 for the cisplatinum treated ventricles. Cisplatinum increased both magnesium efflux and influx and influx in the frog myocardium, increased magnesium content to a higher level and reduced contractile force. The effect of cisplatinum on magnesium transport is attributed to an increase in the charged form of cisplatinum that accumulates inside the cell where chloride content is low and the chloride of cisplatinum is displaced to form a positively charged cisplatinum.

Myocardial magnesium transport: effect of gramicidin S and epinephrine.

Initial magnesium transport in low magnesium Ringer in the frog myocardium consists of at least two systems, a fast system with high capacity with a time constant of 30 seconds and a slow transport system that operates with a time constant of 165 seconds. Both transport systems appear to be electroneutral and can operate either against an electrochemical gradient or with an electrochemical gradient. The fast transport system can transport Mg2+ in an outward direction at 1.84 p mol cm-2 sec-1; the slow system causes Mg2+ to be transported outward at 0.05 p mol cm-2 sec-1. Gramicidin S (5 microM) decreases the slow outward transport system to 0.01 p mol cm-2 sec-1 and at concentrations greater than 1 microM inhibits not only slow magnesium outflux in low calcium Ringer but also inhibits magnesium influx during recovery of Mg2+ in Ringer. Gramicidin S at 5 microM decreases Mg2+ influx from .04 p mol cm-2 sec-1 to 0.01 p mol cm2 sec-1 indicating that influx and efflux may take place on the same transport system. In the presence of 10 mM Mg2+ Gramicidin S increases magnesium content. Epinephrine increases magnesium efflux and overcomes the inhibition of Mg2+ efflux by Gramicidin S.

Methylhistidine Changes in Tibialis Anterior Muscles of 6-Mercaptopurine-Treated Rats

1. Address correspondence to: M.M. Jaweed, Department of Rehabilitation Medicine, Jefferson Medical Center of Thomas Jefferson University, Philadelphia, PA 19107. 2.