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Biomedical subjects

R B Robinson

Publications and source records attributed to R B Robinson.

72 records · Page 4Linked to original sources
PubMed1987

Trophic influence of sympathetic neurons on the cardiac alpha-adrenergic response requires close nerve-muscle association.

In tissue culture, sympathetic neurons exert a trophic effect on myocardial cells to alter the alpha-adrenergic response from positive to negative chronotropism. We hypothesized that this neurotrophic effect might be a result of the release of a humoral substance into the bulk phase of the culture medium by the neurons. The negative chronotropic response to alpha stimulation persisted in nerve-muscle cultures pretreated with the muscarinic antagonist atropine, indicating that acetylcholine is not the trophic agent. Further, variations in the concentration of norepinephrine, epinephrine or dopamine in the nerve-muscle culture media do not account for the presence of a negative chronotropic response to alpha stimulation. Finally, muscle cells grown in the same petri dish with innervated muscle cells, to allow conditioning of the muscle cell environment by the neurons, do not acquire a negative chronotropic response to alpha stimulation. The results of this study clearly demonstrate that this trophic effect is not due to the release of a humoral substance into the bulk phase of the culture medium, but requires close nerve-muscle association. This suggests that the trophic effect may involve localized release and transsynaptic transfer of a chemical substance other than a neurotransmitter.

Acetylcholine↗
PubMed1987

A pertussis toxin substrate regulates alpha 1-adrenergic dependent phosphatidylinositol hydrolysis in cultured rat myocytes.

The chronotropic response of the heart to alpha 1-adrenergic catecholamines influenced by pertussis toxin under certain conditions. In view of the fact that alpha 1-adrenergic action is mediated by the phosphatidylinositol pathway of hormone action in many cells, we examined the hypothesis that alpha-adrenergic agonists stimulate phosphatidylinositol hydrolysis in cardiomyocytes and that this effect is sensitive to pertussis toxin. Addition of norepinephrine to cultured rat ventricular myocytes prelabeled with myo-[2-3H]inositol resulted in rapid and significant accumulation of inositol phosphate (IP1) and inositol biphosphate. Norepinephrine-stimulated IP1 formation was not inhibited by propranolol, but was inhibited by alpha-adrenergic antagonists with an order of potency indicating alpha 1-adrenergic receptor subselectivity: prazosin (alpha 1; 3 nM) greater than yohimbine (alpha 2; 10 microM). The effect of norepinephrine to enhance IP1 formation was markedly attenuated in cells pretreated with pertussis toxin. Pertussis toxin also induced the transfer of ADP-ribose from NAD to a 41,000-dalton membrane protein in these cells. The concentration of pertussis toxin resulting in maximal inhibition of norepinephrine-stimulated IP1 formation correlated well with the concentration of pertussis toxin necessary to completely ADP-ribosylate a 41,000-dalton membrane protein (1 ng/ml). The range over which pertussis toxin inhibited norepinephrine-dependent IP1 formation and ADP-ribosylated the 41,000-dalton substrate was virtually identical. These observations establish a role for a 41,000-dalton pertussis toxin substrate in coupling the alpha 1-adrenergic receptor to phosphoinositol hydrolysis in myocardial cells.

Adenosine Diphosphate Ribose↗
PubMed1986

Regulation of alpha and beta adrenergic receptors by triiodothyronine in cultured rat myocardial cells.

Previously, in this laboratory, we established the presence of alpha and beta adrenergic receptors in primary cultures of neonatal rat heart cells. We now report that exposure to 1-triiodothyronine (10 nM) regulates the binding characteristics of these receptors. As measured by [125I]-I-2-[beta-(4-hydroxyphenyl)ethylaminomethyl]tetralone ([125I]IBE 2254), alpha receptor number (Bmax) decreased by 50% from 37,000 +/- 4,500 to 18,000 +/- 4,300 sites per cell and the equilibrium dissociation constant (KD) decreased from 420 +/- 24 to 150 +/- 37 pM. As measured by [125I]-iodocyanopindolol ([125I]ICYP), beta receptor number increased by 42% from 12,000 +/- 2,600 to 17,000 +/- 4,000 sites per cell with no accompanying change in affinity. An increase in maximal stimulation of adenylate cyclase activity by isoproterenol was also detected under conditions of excess triiodothyronine. No significant changes in agonist or antagonist affinities for either the alpha or the beta adrenergic receptor were detected in thyroid hormone treated cultures. It can be concluded that in cultured myocardial cells thyroid hormone regulates the characteristics of both alpha and beta adrenergic receptors, but in a strikingly different manner.

Adenylyl Cyclases↗
PubMed1985

Acquisition by innervated cardiac myocytes of a pertussis toxin-specific regulatory protein linked to the alpha 1-receptor.

During development, the chronotropic response of rat ventricular myocardium to alpha 1-adrenergic stimulation changes from positive to negative. The alpha 1-agonist phenylephrine increases the rate of contraction of neonatal rat myocytes cultured alone but decreases the rate of contraction when the myocytes are cultured with functional sympathetic neurons. The developmental induction of the inhibitory myocardial response to alpha 1-adrenergic stimulation in intact ventricle and in cultured myocytes was shown to coincide with the functional acquisition of a substrate for pertussis toxin. A 41-kilodalton protein from myocytes cultured with sympathetic neurons and from adult rat myocardium showed, respectively, 2.2- and 16-fold increases in pertussis toxin-associated ADP-ribosylation (ADP, adenosine diphosphate) as compared to controls. In nerve-muscle cultures, inhibition of the actions of this protein by pertussis toxin-specific ADP-ribosylation reversed the mature inhibitory alpha 1-adrenergic response to an immature stimulatory pattern. The results suggest that innervation is associated with the appearance of a functional pertussis toxin substrate by which the alpha 1-adrenergic response becomes linked to a decrease in automaticity.

Adenosine Diphosphate↗
PubMed1985

Modification of Vmax of canine cardiac Purkinje fibers and of the effects of lidocaine by SC-72-14.

We used standard microelectrode techniques to determine the effects of SC-72-14, a monoclonal antibody to the fast sodium channel, on the transmembrane potentials and Vmax of canine cardiac Purkinje fibers, and to study the interactions of the monoclonal antibody with lidocaine. SC-72-14 was prepared with receptor-rich membrane fragments of homogenized eel electroplax as the antigen. It reduced Vmax in a concentration-dependent fashion, and depressed membrane responsiveness, as well. These effects were attributable specifically to SC-72-14, and not to other substances in the vehicle. At slow rates of stimulation, the actions of SC-72-14 and lidocaine on Vmax equaled the sum of their effects, administered alone. These results are consistent with the interpretation that SC-72-14 and the local anesthetic did not compete for the same binding site. In contrast, at fast stimulation rates, the use-dependent blocking effects of lidocaine were suppressed by SC-72-14. The basis for the interference with the use-dependent action of lidocaine is not readily apparent, but may be the result of SC-72-14-induced changes in channel state, receptor affinity for drug, or the availability of ionized drug molecules. These possibilities all await further testing.

Animals↗
PubMed1985

Neuronal regulation of the development of the alpha-adrenergic chronotropic response in the rat heart.

During development, there are changes in the response of automatic cardiac fibers to alpha-adrenergic agonists. In neonatal rat ventricle, in vitro phenylephrine (1 X 10(-8) M) induces an increase in automatic rate from 115 +/- 12 (mean +/- SEM) to 168 +/- 10 beats/min, P less than 0.05. In contrast, in adult rat ventricle, the rate decreases from 36 +/- 8 to 12 +/- 12 beats/min, P less than 0.05. At both ages, the response is attenuated by the alpha 1-antagonist, prazosin (1 X 10(-6) M). We used cultures of neonatal rat myocytes to determine whether maturation of innervation contributes to the ontogeny of this response. All non-innervated cultures showed a positive chronotropic response to alpha-stimulation; phenylephrine (1 X 10(-8) M) increased the rate from 40 +/- 2 to 52 +/- 2 beats/min, P less than 0.05. In contrast, 60% of the myocytes innervated with sympathetic neurons showed a decrease in rate in response to phenylephrine, from 78 +/- 6 to 67 +/- 6 beats/min, P less than 0.05. The negative chronotropic effect of phenylephrine did not result from the release of acetylcholine or adenosine, or the inhibition of presynaptic norepinephrine release by phenylephrine. Furthermore, exposure to neuronal norepinephrine is not responsible for the alteration in muscle cell responsiveness. In conclusion, we have demonstrated the modulation of the myocardial response to alpha-adrenergic stimulation by the occurrence of innervation in tissue culture. This provides an explanation for the previously identified ontogenetic change in alpha-adrenergic effects on intact cardiac fibers from excitation to inhibition.

Adrenergic alpha-Agonists↗
PubMed1984

Preparation and electrophysiological characterization of cardiac cell cultures derived from the fetal canine ventricle.

We have developed a primary cardiac cell culture preparation using the fetal canine ventricle. These cultures exhibited widespread and synchronous spontaneous contractile activity, were sensitive to isoproterenol and had the following transmembrane action potential characteristics (means +/- SE): maximum diastolic potential (MDP) -70.9 +/- 0.9 mV; maximum velocity of phase 0 (Vmax) 84 +/- 4 V/s (and tetrodotoxin sensitive); amplitude (AMP) 98.9 +/- 1.1 mV; duration at half amplitude (APD50) 244 +/- 10 ms at a spontaneous cycle length of 1,600 +/- 100 ms. APD50 was linearly dependent on spontaneous cycle length (CL) (slope = 67 ms/s). The action potential of the intact fetal canine ventricle of comparable gestational age had MDP = -67.0 +/- 1.1 mV, Vmax = 127 +/- 6 V/s, AMP = 92.2 +/- 1.6 mV and APD50 = 153 +/- 2 ms at a paced CL of 500 ms. We conclude that myocardial cultures derived from the fetal dog ventricle exhibit electrophysiological characteristics similar to those of the intact canine ventricle, thereby making them a possible alternative to avian and rodent cardiac cultures.

Action Potentials↗
PubMed1983

Isolated myocytes from adult canine left ventricle: Ca2+ tolerance, electrophysiology, and ultrastructure.

We have developed a method for isolating single cardiac muscle cells in high yield (greater than 5 X 10(7) cells) from the canine left ventricle. Most of the myocytes are single cells with ultrastructural detail indistinguishable from intact ventricular myocardium, and more than 50% of the isolated cells remain elongated for at least 7 h in 0.5 mM calcium. Electrophysiological studies demonstrate that external potassium has a strong influence on repolarization in the isolated ventricular cells. Action potentials in [K+]o = 3.78 mM exhibit a positive over-shoot (greater than zero potential), but repolarization often arrests at congruent to -35 mV unless driven to more negative potentials by hyperpolarizing current. This phenomenon of two levels of resting potential is not observed at [K+]o = 5.78 mM. At the higher potassium concentration, values for maximum diastolic potential, amplitude, maximum rate of rise of phase 0, and action potential duration all are similar to those of intact ventricular muscle. However, the potential at the peak of the action potential plateau (phase 2) in the isolated myocyte is considerably more negative than that of intact myocardium. In addition, there is a conspicuous notch between phases 1 and 2 of the action potential in the isolated myocyte, whereas the notch is small or absent in intact myocardial action potentials. In summary, our method results in a preparation of stable, ultrastructurally and electrophysiologically intact cells, which should prove useful in studies requiring a large and homogeneous population of myocardial cells.

Action Potentials↗
PubMed1979

Correlation of function and morphology of neonatal rat and embryonic chick cultured cardiac and vascular muscle cells.

To develop morphological criteria which can be applied systematically for the identification of isolated cardiac and vascular muscle cells in mammalian and avian primary cultures, we have correlated structural and staining properties with excitability, contraction, and norepinephrine sensitivity of isolated muscle cells. The primary cultures of cardiac and vascular muscle contained muscle cells and nonmuscle cells. The muscle cells could be clearly identified by action potentials, contractility, and Masson's trichrome stain characteristics, similar to those of cells from intact source heart and blood vessels. Furthermore, the muscle cells were highly responsive to norepinephrine, showing unequivocal increases in contraction frequency. The sensitivity to norepinephrine was found to be very high (ED50 = 2.3 X 10(-9) M) Phase-contrast observation was sufficient to identify muscle cells only when those cells were contracting. There were no unequivocal morphological characteristics that distinguished between quiescent muscle cells and nonmuscle cells in the absence of histochemical staining. Ultrastructural examination by scanning electron microscopy failed to distinguish between muscle and nonmuscle cells. Histological staining was, therefore, the only reliable nonfunctional identification process that separated muscle cells from nonmuscle cells. Primary cultures, containing nonmuscle as well as muscle cells, are an important experimental preparation because the cellular heterogeneity probably minimizes muscle cell loss of function and phenotypic changes. The correlation we have established between cell staining and function will facilitate exploration of single cell properties, which together constitute hearts and blood vessels.

Action Potentials↗
PubMed1977

High sensitivity of cultured cardiac muscle cells to autonomic agents.

We have established conditions under which cultured embryonic myocardial cells are highly sensitive to the autonomic agents norepinephrine and acetylcholine and have determined that the most important factors affecting this sensitivity involve the application protocol. Using cells 3-5 days in culture, isolated from ventricles of 13-day chick embryos, the ED50 for noerpinephrine was 800 pM and that for acetylcholine was 370 pM. These cells were more than 2 orders of magnitude more sensitive than 13-day embryonic hearts freshly isolated, but not dispersed. Intracellular recording of the membrane actions of norepinephrine and acetylcholine on these cultured cells showed changes in pacemaker slope similar to those seen in freshly isolated hearts. These data demonstrate that preparation of ventricular muscle as isolated cells in culture does not necessarily result in the loss of sensitivity to autonomic agents. On the contrary, the isolated cells show the highest sensitivity to norepinephrine and acetylcholine that has been reported for the myocardium.

Acetylcholine↗
PubMed1977

Effects of Ca2+ and catecholamines on the guinea pig atrium action potential plateau.

The activation process in isolated electrically driven guinea pig atria was studied by means of simultaneous microelectrode and tension recording. Reducing external calcium from 2.5 to 1.25 mM prolonged the plateau but further reduction of calcium shortened it. Progressively increasing doses of the calcium antagonist D600 (up to 1.4 micrometer), however, monotonically decreased plateau duration. Either protocol monotonically decreased steady-state tension, but with markedly different effects on the restitution relation. Epinephrine, and to a lesser extent isoproterenol, restored plateau duration after exposure to either a calcium-free or D600-containing solution, but only the isoproterenol effect was propranolol sensitive. Addition of calcium chelators enhanced rather than prevented the effect of epinephrine on plateau duration in a calcium-free solution, extending the plateau duration to more than 3 times normal in some cases. These results are explained in terms of two opposing effects of a change in calcium concentration on plateau formation, one action being through the slow inward current and the second through a shift in a calcium dependence of the inward-rectifying, potassium conductance system.

Action Potentials↗
PubMedDate not supplied

G protein-adrenergic interactions in the heart.

Guanine nucleotide binding proteins were examined for their influence in developmental and adaptive models of adrenergic actions in the heart. In primary cultures of rat cardiac myocytes, the positive chronotropic response to the alpha-agonist, phenylephrine, changes to negative when these cells are grown with and innervated by sympathetic nerves from the paravertebral chain. Innervated cells have significantly more G protein, as determined by the ADP-ribosylation reaction catalyzed by pertussis toxin, which is linked functionally to the negative chronotropic response. Adult canine Purkinje fibers that respond to phenylephrine with a decrease in automaticity are also linked biochemically and functionally to a G protein that serves as a pertussis toxin substrate. Fibers that increase in automaticity after exposure to phenylephrine, either under control conditions (a minority of fibers) or after prior exposure to pertussis toxin (a majority of fibers), have markedly reduced levels of G. A G protein was also shown to be important in the blunted adrenergic responsiveness that characterizes congestive heart failure in human subjects. In this model, the receptor complex is beta-adrenergic and the involved G protein is a cholera toxin substrate. Gs is reduced in the lymphocytes of patients with congestive heart failure and increases toward normal after successful therapy. These observations highlight the important roles that G proteins have in adrenergic actions of the heart both with respect developmental and adaptive changes.

Animals↗
PubMedDate not supplied

Effect of cross-transplantation on normotensive and spontaneously hypertensive rat arterial muscle membrane.

Transplantation of arteries into the anterior eye chamber of rats for 8 weeks was used to test the hypothesis that the neurohumoral environment is important in establishing the altered membrane potential (observable during electrogenic ion transport inhibition) of vascular muscle in hypertension. When caudal arteries from 12- to 16-week-old spontaneously hypertensive rats (SHR) or genetically matched Kyoto-Wistar normotensive rats (KNR) were transplanted into the opposite strain, there was no change in the transport inhibited membrane potential (Em) of the arterial muscle cells from that found in freshly excised donor arteries. However, when caudal arteries from 2-week-old animals were transplanted into the anterior eye chamber, the arteries always developed the appropriate Em for the host animal. In other words, a genetically KNR artery developed the Em of an SHR artery in an SHR host; conversely, a genetically SHR artery developed the Em of a KNR artery in the KNR host. These results provide evidence that: 1) the differences between th Em of caudal arteries from SHR and KNR are not inherent in those muscle cells; 2) the change in Em is triggered in young animals preceding development of hypertension, but not after hypertension is established; and 3) the Em alteration of the caudal artery is independent of structural changes that occur in the artery as a result of increased blood pressure (because KNR transplants were not connected in series with the host anterior eye chamber vasculature and subject to the elevated blood pressures). We conclude that the arterial muscle cells up to a certain age respond to an external factor that regulates their Em and presumably their sensitivity to vasopressor agents.

Animals↗