Irreversible Contracture Research Articles

Bone marrow-derived fibroblast migration via periostin causes irreversible arthrogenic contracture after joint immobilization.

Joint contracture causes distressing permanent mobility disorder due to trauma, arthritis, and aging, with no effective treatment available. A principal and irreversible cause of joint contracture has been regarded as the development of joint capsule fibrosis. However, the molecular mechanisms underlying contracture remain unclear. We established a mouse model of knee joint contracture, revealing that fibrosis in joint capsules causes irreversible contracture. RNA-sequencing of contracture capsules demonstrated a marked enrichment of the genes involved in the extracellular region, particularly periostin (Postn). Three-dimensional magnetic resonance imaging and immunohistological analysis of contracture patients revealed posterior joint capsule thickening with abundant type I collagen (Col1a2) and POSTN in humans. Col1a2-GFPTG ; Postn-/- mice and chimeric mice with Col1a2-GFPTG ; tdTomatoTG bone marrow showed fibrosis in joint capsules caused by bone marrow-derived fibroblasts, and POSTN promoted the migration of bone marrow-derived fibroblasts, contributing to fibrosis and contracture. Conversely, POSTN-neutralizing antibody attenuated contracture exacerbation. Our findings identified POSTN as a key inducer of fibroblast migration that exacerbates capsule fibrosis, providing a potential therapeutic strategy for joint contracture.

A novel mouse model of hindlimb joint contracture with 3D-printed casts.

Stiff joints formed after trauma, surgery or immobilization are frustrating for surgeons, therapists and patients alike. Unfortunately, the study of contracture is limited by available animal model systems, which focus on the utilization of larger mammals and joint trauma. Here we describe a novel mouse-based model system for the generation of joint contracture using 3D-printed clamshell casts. With this model system we are able to generate both reversible and irreversible contractures of the knee and ankle. Four- or 8-month-old female mice were casted for either 2 or 3 weeks before liberation. All groups formed measurable contractures of the knee and ankle. Younger mice immobilized for less time formed reversible contractures of the knee and ankle. We were able to generate irreversible contracture with either longer immobilization time or the utilization of older mice. The contracture formation translated into differences in gait, which were detectable using the DigiGait® analysis system. This novel model system provides a higher throughput, lower cost and more powerful tool in studying the molecular and cellular mechanisms considering the large existing pool of transgenic/knockout murine strains.

Ulcerative lupus vulgaris over nose, leading to cosmetic deformity.

Lupus vulgaris (LV), is a chronic and progressive form of secondary cutaneous tuberculosis. In India, it is commonly seen over buttocks, thighs, and legs whereas involvement of nose is quite rare. Ulcerative variant particularly over nose causes destruction of cartilage, leading to irreversible deformities and contracture. High-index of suspicion is required for early diagnosis and prevention of cosmetic deformity. A case of LV over nose in a young male with ulceration is reported who responded well to anti-tubercular therapy, but left with scarring of nose, which could have been prevented if adequate awareness regarding extra-pulmonary cases would have been practiced.

The effect of muscle length and excursion on muscle contracture after tendon injury: A study in rabbit soleus muscles

If tendon repair after injury is delayed, intramuscular fibrosis occurs and finally results in irreversible muscle contracture. The purpose of this study was to determine the effect of preserving muscle length and excursion on the progression of muscle contracture after tendon injury in a rabbit soleus tenotomy model. Forty rabbits underwent tenotomy of the soleus muscles bilaterally and the tendons were managed according to the five experimental groups (N=40). In group A, the tendon was lengthened maintaining half the excursion. In group B, maximal muscle length was preserved and in group C, resting muscle length was maintained. In group D, the tendon was allowed to retract and undergo fibrosis. In group E, the tendon was partially transected and repaired. Four and 8 weeks postoperatively, soleus muscles were harvested from each hind limb and histomorphometric evaluations were performed to measure the connective tissue areas. Electrophysiological studies were carried out to measure the compound muscle action potential to assess the number of functioning muscle fibres. The results showed that maximal muscle length preservation (group B) was the most protective in preventing muscle contracture within 4 weeks of tenotomy, but this effect was gradually offset by prolonged fixation of the muscle, and 8 weeks after tenotomy, maintenance of excursion (group A) was the most protective. These observations may be helpful in the intra-operative evaluation of muscle contracture in neglected tendon ruptures, and could be applied to the management of acute tendon injuries to prevent muscle contracture when immediate anatomical reconstruction is not possible.

Susceptibility to cartap-induced lethal effect and diaphragmatic injury via ocular exposure in rabbits.

Cartap is extensively used to control agricultural pests. Pertinent literatures have indicated that it causes no eye irritation [D.E. Ray, Insecticides derived from plants and other organisms, in: W.J. Hayes, E.R. Laws (Eds.), Handbook of Insecticide Toxicology, Classes of Insecticides, vol. 2, Academic Press, New York, 1991, p. 611; C. Tomlin, Cartap, in: C. Tomlin (Ed.), The Insecticide Manual, 12th ed., British Crop Protection Council, Surrey, UK, 2000, p. 144]; however, the instillation of a little cartap through the eye has caused death in rabbits. The aim of this study was to determine the ocular toxicity of cartap in New Zealand White rabbits. Cartap was directly instilled into the low conjunctival sac of eyes, at doses of 0, 5, 7.5, 10 and 12.5 mg/kg body weight. The changes in the enzymes and isoenzymes of creatine kinase (CK), lactate dehydrogenase (LD), as well as pathological changes in the muscles of the heart, thigh and diaphragm were determined in the cartap-treated rabbits. Moreover, the neuromuscular effect of cartap was examined using the isolated rabbit phrenic-nerve diaphragm model. The results indicated that rabbits developed severe signs and they died within 20 min of ocular instillation. The ocular LD50 of cartap was 8.1 mg/kg body weight. Treatment with cartap increased the activities of CK and LD enzymes and their isoenzymes, CK-1, CK-2, and CK-3 in serum, and CK-3 and LD-5 in the diaphragm. Microscopically, hypercontraction bands and the rupture of myofibers of the diaphragm were observed in dead rabbits. Cartap did not affect nerve-evoked twitch but induced irreversible contracture and twitch depression on the isolated rabbit's diaphragm. These results indicate that the rabbit is susceptible to cartap toxicity; the effect of cartap caused contracture and damage to the diaphragm might play a pivotal role in respiratory paralysis and death of rabbits during intoxication.

Mechanisms of Ca2+ overload induced by extracellular H2O2 in quiescent isolated rat cardiomyocytes.

Rat cardiomyocytes were exposed to H2O2 (1-100 micromol/L) for 10 min with washout for 10 min. Intracellular Ca2+ concentration ([Ca2+]i) was measured using fluo-3. [Ca2+]i increased with 100 micromol/L H2O2 and further increased during washout, causing irreversible contracture in one-half of the cells. The increase in [Ca2+]i with 10 micromol/L H2O2 was modest with few cells showing irreversible contracture and attenuated by caffeine, and [Ca2+]i gradually decreased during washout and this decrease was accelerated by a calcium-free solution, while 1 micromol/L H2O2 did not have any effects on [Ca2+]i or cell viability. Ca2+ overload caused during exposure to 100 micromol/L H2O2 was attenuated by caffeine with improved cellular viability but not by chelerythrine, KB-R7943 or nifedipine. With 100 micromol/L H2O2 calcium-free solution attenuated the increase during exposure and washout while KB-R7943 or chelerythrine partly attenuated further increase during washout but not improved cell viability, but chelerythrine did not have additional effect on calcium-free treatment. Catalase abolished the effects of H2O2. We concluded that the increased [Ca2+]i during exposure to 100 micromol/L H2O2 was caused both by release of Ca2+ from the intracellular store sites including the sarcoplasmic reticulum and by influx through route(s) other than the voltage-dependent Ca2+ channels or Na+/Ca2+ exchanger, although the Na+/Ca2+ exchanger or protein kinase C-mediated mechanism was partly responsible for a further increase during washout.

Effects of Palmitoyl Carnitine on Perfused Heart and Papillary Muscle.

BACKGROUND: Palmitoyl carnitine accumulation during ischemia causes profound electrophysiological changes, resulting in arrhythmias. We studied the electrophysiological and contractile effects of palmitoyl carnitine. METHODS AND RESULTS: Extracellular recordings made by using the endocardial unipolar paced evoked response (PER) in isolated perfused rabbit hearts were compared with action potentials (AP) recorded from septal artery perfused rabbit papillary muscle. Left ventricular pressure was monitored in isolated hearts. In perfused hearts palmitoyl carnitine (30 µmol/L, 30 minutes) significantly (P <.001) increased the latency of activation (St-R interval) by 58% +/- 8% and reduced repolarization time (R-E interval) by 39% +/- 4%. PER duration (St-E interval), was reduced by 30% +/- 3%. Palmitoyl carnitine (30 µmol/L) significantly (P <.001) decreased resting membrane potential (19 +/- 2 mV) of AP, reduced peak amplitude (33.5 +/- 8 mV) and rate of rise of phase 0 (41 +/- 8 V/s). Significant reductions (P <.001) in the action potential duration 50% (129.4 +/- 28 ms) and 90% (139.8 +/- 32 ms) were also observed. An initial positive inotropic effect, which declined as irreversible contracture developed, was also observed. Verapamil (1 µmol/L), nifedipine (1 µmol/L), and caffeine (10 mmol/L) failed to abolish the positive inotropy. CONCLUSIONS: We suggest that palmitoyl carnitine disrupts intracellular calcium homeostasis leading to disturbances in electrical and contractile activity. Its accumulation during myocardial ischemia could contribute to calcium overloading and initiate lethal arrhythmias.

Die Zugschraubenarthrodese von Fingergelenken

Arthrodesis of proximal and distal interphalangeal joints. Joint destruction after primary or secondary arthritis accompanied by activity related pain. Joint instability interfering with pinch and not amenable to soft tissue procedures. Irreversible contracture in flexion or extension. Acute infection. Lack of bone stock. Unusually large medullary canal precluding safe anchorage of the screw. Supine, hand table. Roll of towel under fingers. Tourniquet at upper arm. Elastic band around base of finger in instances of DIP arthrodesis. Regional: plexus- or ring anaesthesia. Arthrodesis using the lagscrew principle. After resection of the destroyed joint surfaces compression of the fragments using screws of the Martin miniset and applying the lagscrew principle. The screw head will be countersunk. Immobilization on a plaster slab supporting forearm, operated and 1 neigh-bouring finger. Physiotherapy. Scew removal is usually not necessary. The screw head sinks into the medullary canal. Lack of sufficient stability. Lack of bony union. Infection. Gaping at site of arthrodesis. Detachment of the extensor tendon. Countersink hole of insufficient size. Between 1982 and 1992 51 patients had 54 lagscrew arthrodeses. 51 arthrodeses in 48 patients could be reexamined at the end of 1992. Average time of follow-up: 3 1/2 years (6 months to 10 years). No postoperative complication was observed and primary fusion was seen in every patient 4 to 13 weeks postoperatively. In 5 instances the screw had to be removed. The angle of arthrodesis varied between 10° and 54°, a vallue determined preoperatively to accommodate the individual needs. The pinch strength reached 75% of that of the finger of the opposite hand in 2/3 of the patients. Symptom-free were 44 patients, 4 had occasional pain (2 had activity related pain of the entire finger and 2 felt pain with changing weather). 86.8% of the patients assessed their result as good or excellent.

The role of the sodium pump and sodium-calcium exchange in recovery from calcium overload in guinea-pig ventricular myocytes.

A marked increase in [Ca2+]i is provoked in isolated guinea-pig ventricular myocytes by return to normal Tyrode solution after a period of exposure to fluid free of divalent cations. When the sarcolemma of the myocyte remains intact, [Ca2+]i falls back towards initial levels over the next 5 to 10 min in spite of the strong irreversible contracture. This recovery fails to occur if either the Na+ pump or Na(+)-Ca2+ exchange is inhibited once [Ca2+]i has risen.

Ca2+ and voltage inactivate Ca2+ channels in guinea-pig ventricular myocytes through independent mechanisms.

1. L-type Ca2+ currents and Ca2+ channel gating currents were studied in isolated guinea-pig ventricular heart cells using the whole-cell patch-clamp technique, in order to investigate the mechanism of Ca(2+)-dependent inactivation. The effect of altering the intracellular Ca2+ concentration ([Ca2+]i) on these currents was studied through photorelease of intracellular Ca2+ ions using the photolabile Ca2+ chelators DM-nitrophen and nitr-5. 2. We found that step increases in [Ca2+]i produced by photorelease could either increase or decrease the L-type Ca2+ current. Specifically, Ca2+ photorelease from DM-nitrophen almost exclusively caused inactivation of the Ca2+ current. In contrast, Ca2+ photorelease from nitr-5 had a biphasic effect: a small, rapid inactivation of the Ca2+ current was followed by a slow potentiation. These two Ca(2+)-dependent processes seemed to differ in their Ca2+ dependence, as small Ca2+ photoreleases elicited potentiation without a preceding inactivation, whereas larger photoreleases elicited both inactivation and potentiation. 3. The mechanism of the Ca(2+)-dependent inactivation of Ca2+ channels was explored by comparing the effects of voltage and photoreleased Ca2+ on the Ca2+ current and the Ca2+ channel gating current. Voltage was found to reduce both the Ca2+ current and the gating current proportionally. However, Ca2+ photorelease from intracellular DM-nitrophen inactivated the Ca2+ current without having any effect on the gating current. 4. The dephosphorylation hypothesis for Ca(2+)-dependent inactivation was tested by applying isoprenaline to the cells before eliciting a maximal rise of [Ca2+]i (maximal flash intensity, zero external [Na+]i). Isoprenaline could completely prevent Ca(2+)-dependent inactivation under these conditions, even when [Ca2+]i rose so high as to cause an irreversible contracture of the cell. 5. We concluded from these experiments that voltage and Ca2+ ions inactivate the L-type Ca2+ channel through separate, independent mechanisms. In addition, we found that Ca(2+)-dependent inactivation does not result in the immobilization of gating charge, and apparently closes the Ca2+ permeation pathway through a mechanism that does not involve the voltage-sensing region of the channel. Furthermore, we found that Ca(2+)-dependent inactivation is entirely sensitive to beta-adrenergic stimulation. These facts suggest that either Ca(2+)-dependent inactivation results from Ca(2+)-dependent dephosphorylation of the Ca2+ channel, or that Ca(2+)-dependent inactivation is modulated by protein kinase A.

The correlation between the increase in slow outward current and in contraction induced by caffeine, ryanodine, and rapid cooling in voltage-clamped frog muscle fibers

The effects of caffeine, ryanodine, and rapid cooling were tested on the depolarization-induced contraction and the apamin-insensitive slow outward current (Iso) of voltage-clamped (double mannitol gap) single frog muscle fibers. Subthreshold caffeine concentrations (0.5-2 mM) induced a monotonic increase in contractile and Iso amplitude. Whatever the concentration, the increase in contraction was roughly twice the one in current. Similar results were obtained upon rapid cooling (20-4 degrees C) in the presence of 0.5 mM caffeine. In the absence of external Na+ (choline-substituted) 10(-5) M ryanodine induced a delayed increase (approximately 30 min) in contraction and in current, shortly before the development of a drastic and irreversible contracture. Here again, the increase in contraction was twice that in current. In the presence of 5 mM tetraethylammonium (TEA) and (or) 25 nM charybdotoxin, 2 mM caffeine still induced a strong facilitating effect on contraction but the parallel increase in current was strongly reduced. The linear relationship between the increase in current and contractile amplitude has a slope approximately 0.5 (whatever the drug used to increase contractility); it is approximately 0.1 in the presence of TEA and (or) charybdotoxin. In conclusion, provided the changes in contractile amplitude are caused by parallel changes in depolarization-induced sarcoplasmic reticulum Ca2+ release, about 50% of the apamin-insensitive Iso is controlled by internal Ca2+ release. The main part of this current corresponds to the TEA- and charybdotoxin-sensitive component of Iso.

Improvement in contractile recovery of isolated rat heart after cardioplegic ischaemic arrest with endogenous phosphocreatine: involvement of antiperoxidative effect?

The aim was to attempt to get further insight into the mechanism of the cardioprotective action of phosphocreatine (PCr). Three experimental protocols were used: (1) The effect was examined of exogenous PCr (10 mmol.litre-1) on myocardial oxidative damage produced by H2O2 perfusion (90 mumol.litre-1) of isolated rat heart. (2) Isolated rat hearts were subjected to 35 min cardioplegic ischaemia followed by reperfusion. A control group was studied along with two PCr groups, one corrected for Ca2+ to compensate its binding with PCr (1.4 mmol.litre-1 CaCl2 in St Thomas's Hospital cardioplegic solution), and the other not (1.2 mmol.litre-1). (3) The effect was studied of PCr alone and in combination with the antioxidant tocopherol phosphate (0.1 mumol.litre-1) on contractile and metabolic recovery of isolated rat heart reperfused after 40 min cardioplegic ischaemia. Studies were performed on hearts of 84 male Wistar rats, weighing 250-300 g. (1) Oxidative stress resulted in irreversible contracture and impairment of sarcolemmal integrity revealed by using the transmembrane tracer ionic lanthanum. These effects coincided with the decrease of developed pressure from 116 (SEM 3) to 38(3) mm Hg and rate-pressure product from 498(13) to 165(16) mm Hg.s-1. The Ca2+ binding property of PCr was estimated experimentally and the stability constant of the complex CaPCr was found to be 35.4(0.7) mmol; from this the Ca2+ bound by PCr was calculated to be 14% in the experimental conditions used. Ca2+ concentration in K-H buffer containing PCr was increased to compensate its binding with PCr. PCr prevented H2O2 induced contracture, preserved sarcolemmal integrity, and attenuated H2O2 induced decrease in developed pressure and rate-pressure product [73(6) mm Hg and 340(28) mm H.s-1, respectively, p less than 0.05 compared with control]. (2) PCr reduced the diastolic pressure [29(10) v 68(10) mm Hg in control group at 30 min of reperfusion, p less than 0.05] and enhanced the developed pressure [81(10) v 46(10) mm Hg in controls, p less than 0.05] and rate-pressure product [325(44) v 158(40) mm Hg.s-1 in controls, p less than 0.05]. When CaCl2 was increased to 1.4 mmol.litre-1 the protective effect of PCr was not abolished. (3) PCr resulted in improvement of developed pressure [49(7) v 18(5) mm Hg in controls at 40 min of reperfusion, p less than 0.05] and rate-pressure product [184(27) v 71(20) mm Hg.s-1 in controls, p less than 0.05]. The degree of contractile recovery in the tocopherol group was almost the same as in the PCr group. Combined addition of PCr and tocopherol further increased the developed pressure and rate-pressure product to 72(4) mm Hg and 284(23) mm Hg.s-1, respectively. Similarly, PCr and tocopherol in combination provided substantial inhibition of creatine kinase release into perfusate, at 3.8(0.4) v 10.9(2.5) IU in controls, p less than 0.05. PCr decreases the vulnerability of myocardium to oxidative stress and ischaemic damage. These effects cannot be explained by PCr induced shifts in Ca2+ concentration. Protective effects of PCr and tocopherol are quantitatively additive, most probably due to their different mechanisms of action, and tocopherol may be effective in extending the ability of PCr to stabilise cell membrane structure.

Effects of nicardipine on myocardial metabolism and coronary haemodynamics: A review

Abstract 1 Nicardipine is a dilator of the coronary vasculature and has a membrane-stabilising effect in ischaemia. In animal models of infarction, it preserved ventricular pump function, and infarct size, wall thinning and left ventricular dilatation were lessened. Pre-treatment before coronary ligation in baboons reduced infarct size. 2 Nicardipine protected myocytes from irreversible contracture when exposed to veratrine and calcium, unlike diltiazem and nifedipine. 3 Intravenous nicardipine increases resting heart rate and mean arterial pressure, and reduces coronary vascular resistance. The coronary vasodilator effects are not affected by β-adrenoceptor blockade. 4 During a cold-pressor test at constant heart rate, arterio-coronary sinus difference in oxygen and the myocardial lactate uptake were improved, along with better indices of left ventricular function. In pacing-stress tests, lactate extraction fraction reduction was improved. 5 An atrial pacing study using MUGA scanning showed improvement or normalisation of ventricular wall motion. 6 In pacing-induced angina, nicardipine reduced lactate production, glucose extraction and glutamate uptake from control levels, reflecting a faster recovery of aerobic metabolism. 7 After PTCA, lactate production was less after intracoronary injection of nicardipine and lactate extraction returned earlier than in controls, indicating a protective effect on the ischaemic myocardium. 8 In a long-term angina study, nicardipine increased exercise duration and reduced ST segment depression at peak exercise. Lactate uptake was increased, the extent being related to plasma levels of nicardipine. Alanine and glutamine production fell, transcardiac release of thromboxane B2 was reduced, and that of 6-keto-PGF1α increased. 9 Nicardipine improves myocardial metabolism in ischaemia, by increasing oxygen delivery in such areas. No negative inotropic effects were noted, rather, left ventricular function improved at rest and during exercise. Afterload reduction is also seen, favouring blood flow redistribution to underperfused areas. Nicardipine may also help maintain the integrity of myocyte membrane in ischaemia.

Suppression of cellular injury during the calcium paradox in rat heart by factors which reduce calcium uptake by mitochondria.

Isolated Langendorff perfused rat hearts were used to study changes in the Ca, Na and K content, contractile force and the loss of cellular material during the Ca paradox. Five minutes perfusion with Ca-free solution containing 1 mM EGTA, followed by 10 min of reperfusion in 1.8 mM Ca causes irreversible contracture, K loss, increase in Na and Ca and a massive release of myoglobin and other cellular material into the perfusate (the calcium paradox). During the Ca-free perfusion the ventricles gain Na but the K content decreases slightly. The size of the Na gain appears to depend upon the buffer used and is larger in bicarbonate than in Tris. When HCO3- or H2PO4- ions are omitted from the bathing solution (in Tris, HEPES, or TES buffered salines) the adverse effects of Ca readmission are reduced. Tris buffer gives the best protection. Metabolic inhibition with FCCP (5 X 10(-7) M), or with CN-(2 X 10(-3) M) together with iodoacetic acid (2 X 10(-3) M), decreases Ca uptake during the Ca paradox and inhibits the release of cellular material. In both cases a contracture is observed. Ruthenium red (10(-4) M) does not inhibit the Ca readmission contracture but reduces the release of cellular material and the gain of Ca and Na. The results suggest that the loss of cellular constituents during the calcium paradox, is related to an active uptake of Ca by the mitochondria and may lead to massive changes in the cellular ion concentration, during Ca-repletion.

Importance of sodium ions in the protective effects of high-potassium, high-glucose solution on electromechanical activities in the guinea-pig myocardium

The protective effects of high-K, high-glucose solution (GK solution) on electromechanical activity in the isolated guinea-pig ventricular muscle were studied. The basal test solution (basal GK solution) contained 30 mM KCl, 230 mM glucose and 4 mM NaHCO3, that is nominally Ca-free. Various types of GK solutions were also prepared by modifying the composition of this solution. When the muscle was exposed to the basal GK solution, the resting potential fell to about -40 mV, the muscle lost the excitability, and an irreversible contracture gradually developed. This contracture was prevented by elevating [Na]o above 60 mM. Reduction of [K]o to 20 mM, addition of EGTA (0.4 mM), or lowering the temperature (23 degrees C) also suppressed the above contracture. When the GK solution with high Na (106-115 mM NaCl added to basal GK solution in exchange for glucose) was applied, contractures frequently developed upon reintroduction of Tyrode solution (Ca-paradox-like phenomenon). Thus the Ca-free GK solution with 60 mM Na (56 mM NaCl added to basal GK solution in exchange for glucose) induced no contracture, either during or after the test period. Recovery of the action potential after this application was all but complete. On the other hand, addition of 0.9 to 1.8 mM Ca to this solution produced another type of contracture which was sensitive to verapamil. The cardioplegic effects of the Ca-free GK solution with 60 mM Na persisted under hypoxic conditions, and glucose appeared to play a significant role in preventing the hypoxia-related contracture. In contrast, the high-Na (110 mM Na) GK solution containing 0.2 to 0.5 mM Ca and Tyrode solution, both of which produced no contracture under normoxic conditions, did produce contractures under hypoxic conditions. Therefore, Ca-free GK solution containing an appropriate concentration (around 60 mM) of Na may protect the normoxic and hypoxic myocardium against intracellular Ca overload. The related mechanisms involved were discussed with special reference to membrane functions and intracellular Ca-regulating systems.

Low resting potentials in single isolated heart cells due to membrane damage by the recording microelectrode

Single myocytes from adult rat hearts were prepared following the method of Powell and co-workers (9, 10, 11). Low resting potentials (Em) could be improved by three techniques. (i) Elevation of Cao to 7.2 mM which, however, mostly resulted in spontaneity and irreversible contracture. (ii) Pre-incubation in a "KB medium" (6). (iii) Use of suction pipettes instead of tapered microelectrodes for intracellular recordings (2). It is concluded that low Em measured previously (11) were due to membrane damage upon microelectrode impalement accompanied by insufficient healing of the membrane around the electrode insertion.

Anoxic injury of adult cardiac myocytes.

Cultured adult cardiocytes were exposed to anoxia. The initial decrease of high-energy phosphates was accompanied by a moderate release of cytosolic enzymes and morphological changes: the appearance of sarcolemmal 'microblebs' (approximately 1 micron in diameter) and an increase of subsarcolemmal vesicles. At ATP levels above 2 mumol/gww, metabolic and morphological alterations were reversible. Probably the sarcolemmal changes are causally related to the loss of macromolecules from reversibly injured cells. At ATP levels below 2 mumol/gww, an increasing number of cells become irreversibly hypercontracted. In these cells cytoplasmic masses are protruded into large 'macroblebs' (10-30 micron in diameter), however sarcolemmal continuity is preserved. Thus, enzyme release, irreversible contracture and cytolysis do not occur simultaneously in anoxic isolated cardiocytes.

Calcium entry blockers inhibit contractures induced by the molluscicide Frescon in Lymnaea stagnalis smooth and cross-striated muscles

The molluscicide Frescon induces irreversible contracture in smooth and cross-striated muscles of the freshwater snail Lymnaea stagnalis. This contracture can be inhibited or reversed by elevated Mg 2+ and reduced Ca 2+ concentrations, by the heavy metals Ni 2+, Co 2+, and Mn 2+, by the rare earth La 3+, and by the calcium channel blocker D-600. The removal of extracellular Ca 2+ also prevents the expression of Frescon action. These results are consistent with the hypothesis that Frescon causes an increase in the Ca 2+ permeability of the sarcolemma.

The ability of isolated myocardial cells of the rat to contract at temperatures near freezing point

1. 1.|Single spontaneously-beating myocardial cells were prepared by enzymatic isolation from rat heart ventricles. Using a TV camera and videotape recorder, beating parameters were characterized with respect to temperature. 2. 2.|The cells obtained showed two types of contractions: (a) action-potential-coupled, fast contractions, which could be triggered electrically but sometimes also occurred spontaneously; and (b) a phasic type of contraction which occurs spontaneously as a slowly (25–200 μm s −1) proceedings wave through the cell and is not coupled to the action potential. 3. 3.|Both the spontaneous phasic and electrically-triggered fast contractions continued down to about 0°C, where the cells went into an irreversible contracture, but recovered if the temperature was raised before the contracture occurred. 4. 4.|In physically-contracting cells, the beat rate and the velocity of the contraction wave were temperature dependent showing a linear relationship on an Arrhenius plot; apparent activation energies were 31 and 54 kJ mol −1, respectively. 5. 5.|When fast contractions were triggered by electric field stimulation, the threshold of cells was reduced as temperature rose. The rheobase values showed that cell excitability decreased more steeply when the temperature fell below 20°C. This change could not be seen in the values of chronaxie, which showed a linear relationship on the Arrhenius plot within the whole temperature range used (6–35°C). 6. 6.|It is concluded that the lower temperature limit is not the same for all structural levels of the rat heart. Myocardial cells are able to contract still at the freezing point while mechanisms initiating the heart beat require higher temperatures to be functional.

Response of bovine intra-ocular muscles to transmural stimulation in the presence of various prostaglandins

Effects of prostaglandins (PGs) E 1, E 2 and F 2α on the electrically stimulated and non-stimulated smooth muscles of bovine intra-ocular tissues were investigated in vitro. PGs E 1, E 2 and F 2α contracted the sphincter and these contractions were not antagonized by either cholinergic or adrenergic blocking agents. PGF 2α was much weaker than PGE 1 or PGE 2. PGs in high concentrations produced an irreversible contracture of the sphincter muscle. In contrast, the dilator muscle contracted with PGF 2α, but not PGE 1 or PGE 2. The ciliary muscle did not respond to these PGs or to indomethacin. Electrical field stimulation with short pulses produced excitation of the intrinsic nerves. The responses produced were not altered in amplitude by the application of any dose of PGs or indomethacin, although indomethacin did lower the tone of the sphincter. PGs did not influence the amplitude of the acetylcholine-induced responses of the intra-ocular muscles. These results indicate that in each of the bovine intra-ocular muscles, PGs play a minor role in neuromuscular transmission both prejunctionally and postjunctionally. Thus, PG-induced contractions of the bovine intra-ocular muscles are considered to occur by a direct action on the muscle rather than on the nerves.