Colocalization of enkephalin-like and choline acetyltransferase-like immunoreactivities in olivocochlear neurons of the guinea pig

The guinea pig lateral superior olive was examined immunocytochemically using antisera against enkephalin and choline acetyltransferase sequentially on the same sections. A colocalization of choline acetyltransferase-like and enkephalin-like immunoreactivities was found in cells of the lateral superior olive that give rise to the lateral system of olivocochlear efferents. Only choline acetyltransferase-like immunoreactivity was observed in the group of olivary nuclei that give rise to the medial group of olivocochlear fibers.     

Distribution of serotonin immunoreactive neurons in the brainstem of the hamster, guinea pig, rabbit, and rat

Immunohistochemical techniques were employed to study the distribution of serotonin (5-HT) immunoreactive neurons in the brainstem of the hamster, guinea pig, rabbit and rat. 5-HT neurons were principally found to be concentrated in the midline raphe nuclei, particularly, the raphe pallidus, raphe obscurus, raphe magnus, raphe median, raphe pontis and raphe dorsalis nuclei. Characteristically, these cell bodies are displayed in bands or wing-like patterns which extend laterally from the raphe into reticular formations. The formations often appear to blend with the catecholamine system. They are particularly evident in the brainstems of the rabbit and hamster which contain wider and more lateral extensions of the serotonergic (5-HT) neurons than those observed in the brainstems of the rat and guinea pig. The widespread distribution of 5-HT immunoreacted cell bodies in the brainstem shows that there are significant prospects of 5-HT in neuronal activities.     

Comparative view of the central organization of afferent and efferent circuitry for the inner ear

In all vertebrates, eighth nerve fibres from the inner ear distribute to target nuclei situated in the dorsolateral wall of the rhombencephalon. In amniotes, primary auditory and vestibular nuclei are readily delineated in that acoustic nuclei lie dorsal and sometimes rostral to vestibular nuclei. Fishes and aquatic amphibians have, in addition to labyrinthine organs, hair cell receptors in the lateral line system. Eighth nerve and lateral line fibres from these sense organs project to the octavolateralis region of the rhombencephalon. In this region, the primary nuclei cannot be easily divided into functionally distinct units. However, modality-specific zones seem to be present for auditory as well as lateral line projections lie dorsal and sometimes rostral to those from vestibular organs. Projections from the primary auditory and vestibular nuclei to higher order centres follow pathways which are conservative in their architecture among vertebrates. Ascending auditory fibres project either directly or via relay nuclei to a large midbrain center, the torus semicircularis (inferior colliculus) and hence to the forebrain. In fishes and aquatic amphibians, the lateral line system also sends a projection to the midbrain and information from this system may be integrated with auditory input at that level. The organization of vestibulospinal and vestibulo-ocular pathways shows little variation throughout vertebrate phylogeny. The sense organs of the inner ear of all vertebrates and of the lateral line system of anamniotes receive an efferent innervation. In anamniotes and some reptiles, the efferent supply originates from a single nucleus (Octavolateralis Efferent Nucleus) while that of "higher" vertebrates arises from separate auditory and vestibular efferent nuclei. The biological significance of this innervation for all vertebrates is not yet understood. However, an important feature common to all is the association of the efferent system with the motor centres of the hindbrain.     

The influence of retinal on complement-dependent immune damage to liposomes

Retinal was incorporated into liposomes containing dipalmitoyllecithin, cholesterol, dicetyl phosphate and galactocerebroside; the latter substance served as antigen. They were compared to control liposomes, lacking retinal, with regard to glucose release due to complement-dependent immune damage in the presence of anticerebroside serum. The liposomes were indistinguishable from each other in the amount of total glucose trapped, light scattering characteristics and phosphate content. The rate and extent of glucose release in 30 min was inhibited by the incorporation of retinal. In addition, inhibition was directyl related to retinal concentration and was also observed in the presence of a wide range of concentrations of antigen and complement. Damage to liposomes in the presence of either guinea pig or human complement was inhibited by retinal; this was in contrast to the erythrocyte system in which the hemolytic activity of guinea pig complement was inhibited while that of human complement was enhanced by retinal. Addition of retinal to performed liposomes did not influence complement-dependent damage. Inhibition occurred only when retinal was present during the initial formation of the model membranes. Inhibition persisted even after washing the liposomes free of any unincorporated retinal. The data indicate that liposomes may be an excellent model for studying the influences of retinal on complement mechanism in membranes.     

Distribution of acetylcholinesterase and monoamine oxidase in the amygdala of the guinea pig

Summary A study of the amygdala of the guinea pig was carried out on material stained by the Nissl, acetylcholinesterase (AChE) and monoamine oxidase (MAO) methods. The material stained for Nissl substance was used primarily as a reference in determining the distribution of the two enzymes. Regional differences in cell size and/or distribution were noted within the lateral, basal, medial and cortical nuclei. In the AChE preparations, it was observed that the large-celled part of the basal nucleus stained very intensely, the small-celled part of the basal nucleus and ventromedial part of the lateral nucleus more moderately, and the dorsolateral part of the lateral nucleus and cortical nucleus lightly. The central and medial nuclei showed almost no reaction. With the MAO method, the greatest staining reaction was seen in the medial nucleus, the medial part of the cortical nucleus, the anterior amygdaloid area and the ventromedial wedge of the putamen adjacent to the central nucleus. In addition, fibres of the stria terminalis stained very darkly.These findings are discussed in relation to the observations of previous authors employing the same methods.Supported in part by the Canadian Medical Research Council Grant No. M.T. 870 and U.S. Public Health Service Grant No. NS-07998. This aid is gratefully acknowledged. We are indebted to Dr. Gorm Danscher for additional material and to Mr. A. Meier, Mrs. L. Munkøe, Mrs. K. Sørensen, Miss M. Sørensen, Miss D. Valgaard, and Miss B. Ørum for skillful assistance.     

Histochemical demonstration of acetylcholinesterase in the hypothalamus of the female guinea-pig

Summary The distribution of acetylcholinesterase (AChE) in the hypothalamus of the female guinea pig has been examined histochemically.Activity was found in neurones of the supraoptic, paraventricular and infundibular nuclei; in the lateral hypothalamic area, in cells dorsomedial to the fornix at the level of the paraventricular nucleus and in a large group of cells surrounding the fornix at the level of the in-fundibular nucleus. A small well-stained group of cells, not identified in histological preparations, was present ventral to the paraventricular nucleus. The neuropil stained at the lateral border of the paraventricular nucleus and in the medial mammillary, suprachiasmatic and dorsomedial nuclei. The walls of some blood vessels stained; activity was particularly strong in vessels in the preoptic area and at the base of the median eminence. Nerve fibres on blood vessels ventral to the hypothalamus also stained.The degree of enzyme activity in the cell groups was compared in immature, pregnant, lactating, ovariectomized and hysterectomized animals. The greatest variation occurred in the infundibular nucleus. Hypophysectomy markedly reduced staining in the supraoptic nucleus. The possibility that AChE may be involved in the elaboration or transport of releasing factors is discussed.The authors are indebted to Dr. J. S. Perry for doing the surgery involved in this work. They are also grateful to him and to Dr. R. B. Heap for helpful discussions and to Miss M. Hamon for excellent technical assistance.     

Experimental uveitis induced by products of activated lymphocytes: Intraocular effects of rhodopsin-induced lymphokines

Immunization of strain 13 guinea pigs by footpad injection of bovine rhodopsin in complete Freund's adjuvant produces experimental autoimmune uveitis (retinopathy) with retinal pathology characterized by destruction of the retinal photoreceptor cell layer, as we reported previously. Since rhodopsin-induced EAU can be passively transferred by immune cells but not antirhodopsin antibody, the present studies were conducted to determine if soluble products of activated lymphocytes (PAL) could cause the retinal pathology seen in experimental uveitis. These studies, reported here, show that intravitreal injection of supernatant factors generated by guinea pig lymph node cells specifically activated in vitro with rhodopsin or purified protein derivatives of tuberculin or nonspecifically activated with the mitogen concanavalin A produce uveitis in the normal guinea pig eye. The PAL produced mononuclear cell infiltration in the vitreous and a retinopathy with loss of retinal photoreceptor cells. Inflammatory cell infiltrates were found in the retina but not found in the anterior segment of the eye. Control lymphocyte supernatants did not produce retinal pathology. In guinea pigs sensitized to rhodopsin-complete Freund's adjuvant, intravitreal injection of the PAL produced a mononuclear vitreal infiltrate, disruption of the photoreceptor cell layer, necrosis of the inner retina, and a granulomatous infiltrate in the choroid with damage to the retinal pigment epithelium. Injection of control supernatants into the rhodopsin-complete Freund's adjuvant sensitized guinea pig eye did not produce inflammation and the retinal photoreceptor cell layer remained intact. Results from our studies indicate a role for the PAL along with other, yet undefined, factors in the initiation of autoimmune uveitis and in the autoimmune pathology of the retina.     

Thyrotropin-Releasing Hormone Receptor Binding Sites: Autoradiographic Distribution in the Rat and Guinea Pig Brain

Thyrotropin-releasing hormone (TRH) binding sites were labeled in vitro in mounted brain tissue sections from rat and guinea pig brains with [3H]methyl TRH and localized autoradiographically using 3H-sensitive film. Regional densities of TRH binding sites were measured by computer-assisted microdensitometry. The distribution of sites in both species was highly heterogeneous. In both guinea pig and rat brains, the highest densities of binding sites were seen in the amygdaloid nuclei and the perirhinal cortex. In contrast, in other brain areas, a clear difference between the distribution of sites in rat and guinea pig was found. The temporal cortex, pontine nuclei, and interpeduncular nucleus, which contained high densities of binding in the guinea pig, were scarcely labeled in the rat. The accessory olfactory bulb and the septohippocampal area presented in the rat higher concentrations of binding sites than in the guinea pig. Other brain areas showing intermediate to low densities in both species were accumbens nucleus, bed nucleus of the stria terminalis, dentate gyrus, facial and hypoglossal nuclei, and gelatinosus subnucleus of the trigeminal nerve, among others. The anterior pituitary also presented low to intermediate concentrations of receptors. The distribution of TRH sites here described does not completely correlate with that of endogenous TRH, but is in good agreement with previous biochemical data. The results are discussed in correlation to the physiological effects that appear to be mediated by TRH.     

Golgi studies of the first optic ganglion of the ant,Cataglyphis bicolor

The neurons of the first optic ganglion (the lamina) in the desert ant, Cataglyphis bicolor, have been studied with the light microscope after Golgi silver impregnation. The different types of retinal and laminal fibres and their configuration are compared with the results obtained in the bee. The first synaptic region in the visual system of the ant lies proximally to the fenestrated layer below the basement membrane and the layer containing the monopolar cell bodies. The synaptic region can be separated into three morphologically different zones: (1) The most distal layer where the short visual fibres end at two different levels. The short visual fibres and some laminal fibres (monopolar cell fibres) also show lateral elements in this region. (2) The second layer appears almost free of branches of retinal and laminal fibres. (3) The most proximal layer, which has a characteristically dense horizontal structure resulting from the lateral elements of long visual, centrifugal, monopolar and tangential fibres. Nine cell axons arising from each ommatidium leave the retina. Six of these are short visual fibres and end at two different levels in the lamina. Three different types of short visual fibres can be distinguished by their different terminal depths and lateral branching pattern. The remaining three fibres, the long visual fibres, terminate in the medulla. They can be distinguished from each other by their lateral elements in the lamina neuropile. The five morphologically different laminal fibre types (axons of the monopolar cells in the lamina) have different shapes and different arborizations at different levels. Tangential, centrifugal and incerta sedis-fibres, which originate either from cell bodies in the cell body layer at the periphery of the outer chiasma or more centrally, terminate in the synaptic region of the lamina. Consideration is given to the clearly demarkated arrangement and length of the branching pattern of retinal and laminal fibres at different levels of the synaptic region of the lamina. In addition, a hypothetical connectivity pattern is discussed.     

Histological pictures of muscles and an evaluation of cellular infiltrations in human polymyositis/dermatomyositis,as compared to the findings in experimental Guinea pig myositis

We tested whether intramuscular injections of dermatomyositis (DM) patients' sera into guinea pig muscles can be used to transfer myositic alterations to these animals. Additionally, similar tests were performed using neoplastic patients' sera and sera from non-neoplastic, non-myositic patients. The DM patients' sera induced idiopathic inflammatory myopathy (IIM) type histological changes in muscle fibres in guinea pig quadriceps muscles, which were especially evident 72 h after sera injections. Immunohistochemical stainings of myositic guinea pig muscles were done for guinea pig pan-T-cells, monocytes/macrophages, the neuronal marker-protein gene product 9.5 (PGP 9.5) and protein S-100. Our studies proved that the factor(s) responsible for the appearance of characteristic alterations in diseased muscles during the course of DM is/are present in patient sera.     

Neurokinin A in cerebral vessels: characterization, localization and effects in vitro

Nerve fibres displaying neurokinin A (NKA)-immunoreactivity (IR) were seen in trigeminal nerve cell bodies and around cerebral blood vessels. NKA-positive fibres had the same general distribution as those displaying substance P (SP)-IR. Double or sequential immunostaining revealed coexistence of NKA- and SP-IR in a population of small nerve cell bodies in the trigeminal ganglion and in perivascular nerve fibres of brain vessels; both tachykinins were also noted to coexist with calcitonin gene-related peptide (CGRP)-IR. The presence of NKA- and SP-IR in cerebral vessels from guinea pig was verified by high-performance liquid chromatography and radioimmunochemistry. The levels NKA-IR were higher than those of SP-IR in cerebral vessels of rat, guinea pig and rabbit. In cat, pig, cow and human brain vessels, the levels of NKA- and SP-IR were equal. Major cerebral vessels at the base of the brain contained higher levels of NKA- and SP-IR than pial vessels on the cerebral convexities. Only low levels of NKA-IR and SP-IR were measured in choroid plexus and dura mater. Precontracted isolated arterial segments of middle cerebral (cat), basilar (rabbit, guinea pig and rat) and pial arteries (man) relaxed following the in vitro administration of NKA and SP. The responses occurred in the same concentration range; the IC50 value for NKA was, however, about 10 times higher than that for SP, while the maximum relaxation was equal. In basilar arteries from guinea pig, the peptides NKA, SP and CGRP all induced strong and potent relaxations. There was no evidence that one of the peptides might potentiate the relaxant effects in vitro of another. The present data suggest that NKA, SP and CGRP are costored and can be released together and cooperate in the mediation of vascular reactions in response to activation of the trigemino-cerebrovascular pathway.     

The intermediate filament complement of the retina: a comparison between different mammalian species

We compared the intermediate filament expression of the various cell types in the fully differentiated neural retina from rat, mouse, rabbit, guinea pig, cow, pig, and cat. Many cell types had an intermediate filament complement conserved across species boundaries, such as Müller cells and retinal ganglion cells. In some species (rabbit, guinea pig, and cow), however, we were unable to visualize GFA (glial fibrillary acidic)-positive retinal astrocytes, although such profiles were clearly visible in the remainder. Horizontal cell staining proved to be extremely species-variable. In rat and mouse the processes of these cells were identically displayed with antibodies to vimentin and all three neurofilament triplet proteins. In cow they decorated with antibodies to vimentin and antibodies to the two lower molecular weight neurofilament proteins alone, whereas in pig, rabbit and guinea pig all three neurofilament proteins but not vimentin were present. Finally cat horizontal cells stained for all three neurofilament proteins, some finer processes being additionally stainable with vimentin. A further surprise was the visualization of profiles positive only for the two lower molecular weight neurofilament proteins in the inner nuclear layer of both rabbit and guinea pig retina but not the other species. The implications of these results will be discussed.     

Inter- and intraspecies comparisons of fibre type distribution and of succinate dehydrogenase activity in type I, IIA and IIB fibres of mammalian diaphragms

Fibre types in the costal region of the diaphragm muscle of several mammalian species with widely different respiratory rates were examined microphotometrically for succinate dehydrogenase (SDH) activity. Mean activities indicated no significant (p greater than 0.05) difference between the type I and IIA fibres for any of the species examined. SDH activities in type IIB fibres were significantly lower (p less than 0.05) than either the type I or type IIA fibres in the cat, guinea pig, rat and rabbit whereas in the mouse no difference was found. The dog had no classical type 1B fibres. Analysis of the distribution of SDH activities by fibre type indicated a wide scattering of scores with no distinct separation between fibre types. Large differences in SDH activity were noted between species. Mean SDH activities were highest in the mouse and rat, intermediate in the rabbit and guinea pig and lowest in the cat and dog. These data suggest an association between respiratory rate and aerobic oxidative potential of the various fibre types in diaphragms of the species examined.     

Synthesis and amino acid composition of basic proteins in mammalian sperm nuclei.

The basic chromosomal proteins (SCP) of human, mouse, rabbit and guinea pig sperm nuclei were characterized by polyacrylamide gel electrophoresis and amino acid analysis. Spermatozoa were decapitated with 1% SDS and the nuclei recovered by density gradient centrifugation. Examination by Nomarski and electron microscopy revealed the nuclei to be intact and 99% pure. The basic proteins were extracted from nuclei, aminoethylated and purified by ion exchange chromatography and gel filtration chromatography.The SCP of human, rabbit and guinea pig gave single protein bands with similar mobilities when subjected to polyacrylamide gel electrophoresis. In contrast, aminoethylated mouse SCP consisted of two proteins, SCP·AE₁ and SCP·AE₂, which had different electrophoretic mobilities. The SCP of these mammalian species were characteristically rich in arginine (47–54.4%) and cysteine (7.7–12.2%). Major differences existed in the amino acid compositions of these proteins. Mouse and human SCP were rich in histidine (12.2 and 7.7%, respectively) and guinea pig was high in tyrosine (11.7%) and phenylalanine (3.5%). Valine was detected only in rabbit SCP and proline in human and guinea pig. Aspartic acid, methionine and tryptophan were not detected in all four species. Studies on the incorporation of [³H]arginine into mouse SCP demonstrated that these basic proteins are synthesized during the terminal stages of spermatogenesis and are subsequently conserved.     

Inter- and intraspecies comparisons of fibre type distribution and of succinate dehydrogenase activity in type I,IIA and IIB fibres of mammalian diaphragms

Summary Fibre types in the costal region of the diaphragm muscle of several mammalian species with widely different respiratory rates were examined microphotometrically for succinate dehydrogenase (SDH) activity. Mean activities indicated no significant (p>0.05) difference between the type I and IIA fibres for any of the species examined. SDH activities in type IIB fibres were significantly lower (p<0.05) than either the type I or type IIA fibres in the cat, guinea pig, rat and rabbit whereas in the mouse no difference was found. The dog had no classical type 1B fibres. Analysis of the distribution of SDH activities by fibre type indicated a wide scattering of scores with no distinct separation between fibre types. Large differences in SDH activity were noted between species. Mean SDH activities were highest in the mouse and rat, intermediate in the rabbit and guinea pig and lowest in the cat and dog. These data suggest an association between respiratory rate and aerobic oxidative potential of the various fibre types in diaphragms of the species examined.     

Neuropeptide Y-like immunoreactivity in intramural ganglia of the newborn guinea pig urinary bladder

Immunoreactive neuropeptide Y (NPY) was demonstrated in neuronal elements in the urinary bladder wall of the newborn guinea pig. Numerous intramural ganglia were found lying among the smooth muscle bundles and in the submucosa, and NPY-like immunoreactive nerve cell bodies were demonstrated within all of these ganglia. Nerve fibres containing NPY were also richly distributed in the detrusor muscle, submucosa and around blood vessels. In dissociated cell cultures from newborn guinea pig detrusor muscle, a subpopulation (70-85%) of both mononucleate and binucleate intramural neurones was shown to contain NPY-like immunoreactivity. A low percentage (1-6%) of the intramural bladder neurones contained dopamine-beta-hydroxylase. In conclusion, while some NPY-containing nerve fibres in the wall of the bladder are of sympathetic origin, especially those supplying blood vessels, the results of this present study establish that many of these NPY-containing nerve fibres originate from non-adrenergic cell bodies within the intramural bladder ganglia.     

A comparison of two ATPase based schemes for histochemical muscle fibre typing in various mammals

A comparative study was performed on the fibre populations in tibialis anterior muscles of mouse, rat, guinea pig, rabbit, cat and dog using the two different methods of histochemical staining for myofibrillar ATPase after acid (Brooke and Kaiser 1970) or alkaline preincubations (Guth and Samaha 1970). For all species a complete correspondence existed between type I (Brooke and Kaiser 1970) and beta fibres (Samaha et al. 1970). Gross correspondence (greater than 85%) existed between IIA and IIB (Brooke and Kaiser 1970) and alpha beta and alpha fibres (Samaha et al. 1970) respectively in mouse, guinea pig, rabbit, cat and dog. In the case of mouse and dog, this high degree of correspondence was based on the assumption that mouse tibialis anterior contains no type I and the dog no type IIB fibres. For the rat, a pronounced overlap existed between IIA fibres on the one hand and alpha beta and alpha fibres on the other hand as well as between IIB fibres and alpha beta and alpha fibres. These observations lead to the conclusion that the two classification schemes are not interchangeable for all species and that the two terminologies should be used only in relation with the methods from which they were derived.     

Immunohistochemistry of biogenic polypeptides in nerve cells and fibres of the guinea pig inferior mesenteric ganglion after perturbations

Immunohistochemistry of peptide- and dopamine-beta-hydroxylase-(DBH)-containing varicose nerve fibres and ganglion cells, respectively, in the guinea pig inferior mesenteric ganglion was investigated following a) transsection of mesenteric (colonic) branches, b) transsection of central (lumbar splanchnic, intermesenteric and hypogastric) branches, and c) transplantation into the spleen. The findings indicate that pathways of different opioid peptides are not identical. Met-enkephalin- and met-enkephalin-arg-phe- (cleavage products from pre-proenkephalin) containing fibres course in central branches to make contact in the inferior mesenteric ganglion. Dynorphin- and alpha-neo-endorphin- (deriving from pre-prodynorphin) containing fibres as well as leu-enkephalin- (included in the dynorphin sequence) fibres appear to rise not only from central and from enteric somata, but also from intraganglionic noradrenergic neurons. Similar pathways seem to be used by VIP- and by neurotensin-immunoreactive fibres, although intraganglionic neurotensin-immunoreactive cell bodies are rare. Practically all substance P- and most CGRP-immunoreactive fibres enter the ganglion via central branches and, to a large extent, traverse it, but some CGRP-immunoreactive influx appears to come from the intestine. The origin of intraganglionic substance P- and CGRP-immunoreactive fibres after ganglion transplantation remained unidentified. Somatostatin- and neuropeptide Y-immunoreactive fibres predominantly have an intraganglionic origin as have DBH-immunoreactive noradrenergic fibres. The demonstrated alterations in neuropeptide immunoreactivity of intraganglionic and periganglionic nerve fibres following the applied transsection procedures contribute to the present knowledge on origin and destination of peptidergic transmitter segments in the guinea pig inferior mesenteric ganglion. Moreover, the present study provides evidence that intrinsic participation in intraganglionic fibre supply is more extensive than hitherto believed.     

The efferent connections of the lateral septal nucleus in the guinea pig: projections to the diencephalon and brainstem

Summary The anterograde Phaseolus vulgaris-leucoagglutinin (PHA-L) tracing technique was used to determine the distribution of efferent fibers originating in the lateral septal nucleus of the guinea pig. For complementary detection of the chemical identity of the target neurons, double-labeling immunocytochemistry was performed with antibodies to PHA-L and to vasopressin, oxytocin, vasoactive intestinal polypeptide, serotonin or dopamine -hydroxylase, respectively. The hypothalamus received the majority of the PHA-L-stained septofugal fibers. Here, a specific topography was observed. (1) The medial and lateral preoptic area, (2) the anterior, lateral, dorsal, posterior hypothalamic and retrochiasmatic area, (3) the supraoptic, paraventricular, suprachiasmatic, dorsomedial, caudal ventromedial and arcuate nuclei, and (4) the tuberomammillary, medial and lateral supramammillary, dorsal and ventral premammillary nuclei always contained PHA-L-labeled fibers. The rostral portion of the ventromedial nucleus and the medial and lateral mammillary nucleus only occasionally showed weak terminal labeling. In other diencephalic areas, termination of PHA-L-labeled fibers was observed in the epithalamus and the nuclei of the midline region of the thalamus. In the mesencephalon, terminal varicosities occurred in the ventral tegmental area, interfascicular and interpeduncular nucleus, and periaqueductal gray. In addition, the dorsal and medial raphe nuclei of the metencephalon, together with the locus coeruleus and the dorsal tegmental nucleus, received lateral septal efferents.     

Effect of ouabain on mechanical and electrical properties of rat and guinea pig hearts at 180 C.

The effects of ouabain 10(-6) M on rat and guinea pig hearts have been studied at 18 degrees C, in order to reduce almost fully both the Na+, K+-dependent ATPase activity and the ouabain induced inhibition of this enzyme. In isolated guinea pig hearts the positive inotropic response to ouabain obtained at 32 degrees C disappeared at 18 degrees C. On the contrary, the contractile strength of rat hearts was slightly reduced by ouabain and in the same manner at both temperatures. Current and voltage clamp experiments carried out at 18 degrees C in ventricular fibres revealed that ouabain 10(-6) M decreased both the action potential overshoot and the fast sodium current in rat and guinea pig, by reduction of the membrane sodium conductance. Ouabain did not change the calcium current in guinea pig preparations, whereas in rat heart muscle this current was reduced. The effects of ouabain on both the action potential plateau and outward repolarizing current indicated some inconsistencies from preparation to preparation and cannot therefore be considered as significant. The persistence of the ouabain induced alterations of g Na (in rat and guinea pig) and calcium current (in rat) at 18 degrees C supports the hypothesis of two ouabain cell receptors in heart muscle.