Life Sciences, Vol . 22, pp . 1223-1228 Printed in the II .S .A .
Pergamon Preee
THE TROPHIC INFLUENCE OF AUTONOMIC NERVES ON ELECTRICAL PROPERTIES OF THE CELL MEMBRANE IN SMOOTH MUSCLE William W . Flaming Department of Pharmacology West Virginia University Medical Center Morgantown, West Virginia
26506
Summary The relationship of neurotransmission to sensitivity in a variety of effector cell types is briefly surveyed . The several cellular changes which contribute to supersensitivity in dener vated skeletal muscle are listed . Subsequently evidence is reviewed regarding the role of a partial depolarization of the smooth muscle cells in the development of supersensitivity in the guinea-pig vas deferens . The depolarization appears to be the result of the decreased activity of an electrogenic Na+, K+ pump . The applicability of these findings to other autonomically innervated effectors is discussed . An intriguing relationship which is frequently manifest in research in the neurnsciences is the trophic relationship between nerves and effector cells . Not only do nerves demonstrate obvious moment-to-nroment regulation of effector cell activity, but they also have a long-term influence over many physiological and biochemical characteristics of the effector cells . Pharmacologic agents have provided a rich source of tools with which to study trophic relationships and, hence, pharmacologists have had a particular interest in these relationships . Our own laboratory has had a primary orientation toward the neurotrophic regulation of smooth muscle as manifest by enhanced sensitivity of the muscle to agonists consequent to chronic depression of neurotransmission . The phenomenon of denervation supersensitivity has been recognized for over one hundred years and has been the subject of considerable research (1) . However, the cellular mechanisms responsible for supersensitivity have been elusive, the more so because of the tendency of investigators to seek a single mechanism to explain the phenomenon . The first mechanism to be clearly identified occurs in skeletal muscle and was recognized through experiments which "mapped" the cholinoceptors on single muscle fibers by means of microelectrodes and microiontophoresis (2) . Such experiments led to the hypothesis that denervation supersensitivity in skeletal muscle is the result of a "spread" of receptors outward from the endplate . The validity of this hypothesis has subsequently become firmly supported by studies with the highly specific cholinoceptor-binding substance, 0300-9653/79/0410-1223$02 .00/0
Çopyright © 1978 Pergamon Press
1224
Neurotrophic Influence on Smooth Muscle
Vol . 22, No .e 13-15, 1978
a-bungerotoxin (3) . It is important to emphasize, however, that recent evidence clearly indicates that several different cellular mechanisms contribute to supersensitivity in skeletal muscle . These include receptor spread, complex electrophysiologic changes, altered calcium binding and a decrease in cholinesterase activity (4,5) . Interest in supersensitivity has gained renewed momentum in recent years with the recognition that denervation supersensitivity is an expression of a cellular homeostasis common to many types of excitable cells . The cells have the capacity to accomodate to chronic changes in the stimulus they receive and/or their own level of activity (6) . Excitable tissues in which supersensitivity has been demonstrated, include skeletal muscle, smooth muscle, cardiac muscle, exocrine glands, central neurons and the pineal gland . Given the evidence that multiple mechanisms contribute to supersensitivity in skeletal muscle and the physiologic diversity of the cells which can become supersensitive, it is obviously risky to assume that all of the same mechanisms contribute to supersensitivity in all types of cells . A major step in identifying the mechanisms responsible for supersensitivity in smooth muscle occurred when Trendelenburg (7) established that autonomic denervation produced two entirely separable types of supersensitivity . One type is consequent to inhibition or degeneration of a site of loss of a drug, such that a larger portion of the administered drug reaches the site of action . The sensitivity of the responding cells is not changed, only the distribution and/or fate of the agonist in the tissue . Examples are loss of adrenergic neuronal uptake (8) or extraneuronal uptake (9) . The other type of supersensitivity has been termed disuse, postsynaptic, postjunctional, or nondeviation supersensitivity (10) and represents an actual change in the sensitivity of the responding cells . It is this second type of supersensitivity which is under investigation in our laboratory and is the subject of this review . Postjunctional or nondeviation supersensitivity develops in many types of excitable cells whenever normal neuroeffector transmission is chronically and markedly depressed . The result is independent of the means used to inter rupt transmission (6) . In smooth muscle the supersensitivity is characterized by a delayed onset and nonspecificity, that is, the sensitivity is increased to a similar degree to several unrelated agonists . For example, in the guineapig vas deferens, depression of adrenergic transmission can be achieved by postganglionic denervation ("denervation"), preganglionic denervation ("decentralization") or chronic administration of reserpine (11,12) . Supersensitivity appears and is fully developed four days after impairment of transmission . The dose-response curves for norepinephrine, acetylcholine, histamine and potassium are all shifted to the left by a mean factor of 2 to 4 (12) . The nonspecificity of supersensitivity in smooth muscle led to the conclusion that neither a qualitative nor a quantitative change in receptors is likely to be the primary mechanism for the sensitivity change (13) . Subse quently, indirect evidence led to the hypothesis that a partial depolarization contributes to supersensitivity in smooth muscle (14) . More recent work in our laboratory has been aimed at directly measuring electrophysiologic and biochemical changes in supersensitive smooth muscle, particularly that of the guinea-pig vas deferens . Intracellular recording established that the membrane potential is reduced by 8-10 mV by chronic denervation or decentralization of the vas deferens . The time-course of the depolarization is the same as the time-course of the appearance of the supersensitivity (15) . Of equal significance, the threshold level at which action
Vol . 22, No .s 13-15, 1978
Neurotrophic Influence on Smooth Muscle
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potentials are elicited is unchanged (16) . Thus, the average amount of depolarization necessary to induce contraction is reduced by 35% by denervation . Consequently, a smaller concentration of a depolarizing agonist would be required to produce contraction . If, as the above results suggest, a partial depolarization contributes to the increased sensitivity of the smooth muscle cells, any procedure which causes an acute depolarization of control cells should also render them supersensitive . There are several means by which the smooth muscle cells of the guinea-pig vas deferens may be depolarized, some of them dependent upon the fact that, in this organ, the Na+, K+ pump of the smooth muscle cells is electrogenic . If a Na+ , K+ pump is electrogenic, it transports ions unequally across the membrane, thus creating a current and directly contributing to the membrane potential . According to Droogmans and Casteels (17), the total membrane potential of a cell with an electrogenic pump has two components as follows : in which Em E d i tôtal membrâne potential, Ediff = diffusion potential, Rm = membrane resistance and i p = the electrogenic current . Ouabain, a specific inhibitor of the Na+, K+ pump, reduces Em by reducing ip . ChanQing the concentration of external potassium ([K+] ) can affect either Ediff lbY changing the concentration radient across tRe membrane) or i (by inhibiting or stimulating the Na , K+ pump) . The relationship of [~+] o to these factors in the guinea-pig vas deferens is such that either reducing the [K+] o from 5 .8 mM (control solution) to 2 .9 mM or increasing it from 5 .8 to 11 .6 mM causes a partial depolarization of the membrane . The effects of ouabain, low and high [K+] o on resting potential were tested with microelectrodes in paired vasa deferentia . Other pairs of vasa deferentia were used to determine the effects on dose-response curves of histamine and methoxamine (chosen instead of norepinephrine because its doseresponse curve is unaffected by alterations in adrenergic neuronal uptake) . Ouabain (10 - 5M), half normal [K+] o and twice normal [K+] o each caused a depolarization of 8-10 mV and increases of 2 .5- to 5-fold in sensitivity to histamine and methoxamine (18,19) . These results compare extremely well with decentralization and denervation which cause a depolarization of 8-10 mV and increases in sensitivity of 2- to 4- fold . It is concluded that the depolarization produced by denervation and decentralization is adequate to produce the supersensitivity which accompanies it . The amount of ouabain used in the above experiments, 10'SM, is approximately optimal for the inhibition of Na+, K+ ATPase in the vas deferens (unpublished observations) . The fact that it produces a depolarization similar in magnitude to that produced by decentralization and denervation suggests the possibility that the latter procedures may also inactivate the electrogenic component of the Na+, K+ pump . Support for this suggestion comes from the fact that ouabain has no effect whatsoever on the membrane potential or the sensitivity of chronically denervated vasa deferentia (unpublished observations) . Experiments were therefore carried out to determine if there were any differences between control and supersensitive guinea-pig vasa deferentia in the activity of ouabain-sensitive Na+, K+ ATPase . Chronic denervation, decentralization or reserpine pretreatment decreased the activity of the enzyme 25-50% (20,21 and unpublished observations) . Kinetic analyses have established that the decreased activity is associated with a decreased Vmax ; Kg is unchanged (unpublished observations) .
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Neurotrophic Influence on Smooth Muscla
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The evidence outlined above leads to the conclûsion that a partial depolarization of the smooth muscle membrane is an important contributing factor to supersensitivity in the guinea-pig vas deferens . It should be noted, how ever, that there is at least one other contributing factor . Westfall (22) has shown that there is still a difference in sensitivity to agonists between control and denervated vasa deferentia when they are completely depolarized . Westfall suggested that this second factor involves an alteration in calcium mobilization . There is evidence in vascular smooth muscle that calcium does playa role in supersensitivity (23,24) . An important question arises . Is the guinea-pig vas deferens typical of other smooth muscles in regard to the electrophysiologic contribution to supersensitivity? No other smooth muscle has been studied as thoroughly in this regard . However, there is some evidence for a role of membrane potential in supersensitivity in the guinea-pig ileum (14), the rabbit saphenous artery (25), the rat portal vein (26) and even in cardiac muscle (27) . The role of an electrogenic pump has not been investigated as yet in any of these except the saphenous artery . Our preliminary evidence in that organ is that an electrogenic pump may be involved (unpublished observations) . An interesting contrast is the rat vas deferens . The smooth muscle of that organ does not appear to have an electrogenic pump and does not undergo partial depolarization in association with denervation supersensitivity (28) . Instead, there is a change in the threshold for electrical activation . Thus, even in the rat vas deferens, membrane electrical properties do seem to contribute to supersensitivity . References 1 . W .B . CANNON and A . ROSENBLUETH, The Su ersensitivit of Denervated Structures, Macmillan, New York 949 . 2 . S . THE LE F, Physiol . Rev . 40 734-152 (1960) . 3 . S . THESLEFF, Ann . N .Y .~Icad.Sci . 228 89-103 (1974) . 4 . W .W . FLEMING,Rev . Neurost . X43-x(1976) . 5 . M .G . McCONNELLând L .L . SIMPSON, _J . Pharmacol . E~. Ther . 198 507-517 (1916) . 6 . W .W . FLEMING, J .J . McPHILLIPS and D .P . WESTFALL, Rev . P siol . _68 55-119 (1973) . 7 . U . TRENDELENBURG, Pharmacol . Rev . 15 225-276 (1963) . 8 . U . TRENDELENBURG, a~ rm~. Rév . T$ 629-640 (1966) . 9 . U . TRENDELENBURG and K .-H . GRAÉFE, Proc . _Fed . Amer . Soc . Ex~. Biol . _34 1971-1974 (1975) . 10 . W .W . FLEMING, Proc . Fed . Amer . Soc . ~Ex . Biol . 34 1969-1970 (1975) . Pharmacy 11 . D .P . WESTFALL, Brit . 39 110-1 (~0) . 12 . D .P . WESTFALL, D .C . McCLURE and W .W.FLEMING, J . Pharmacol . E~. Ther . 181 328-338 (1972) .. 13 . h. HUDGINS and W .W . FLEMING, J . Pharmacol . E~. Ther . 153 70-80 (1966) . 14 . W .W . FLEMING, J . Pharmacol . Exp. Ther . 1277 - 85 1968 15 . W .W . FLEMING and D .P . WESTFALL, JP~armâcol . E~. Ther . 192 381-389 . (1975) . 16 . K . GOTO, D .P . WESTFALL and W .W . FLEMING, _J . Pharmacol . E~ . Ther . (in press) . 17 . G . DROOGMANS and R . CASTEELS, P siolo of Smooth Muscle, p . 11, ed . E . Bülbring and M .F . Shuba, Raven ress, New Yorc 9 18 . P .R . URQUILLA and W .W . FLEMING, Pharmacol ogist 16 273 (1974) . 19 . P .R . URQUILLA and W .W . FLEMING, Proc . Fee ~. Amer . Soc . Ex~. Biol . 34 818 (1975) .
Vol . 22, No .s 13-15, 1978
Neurotrophic Tnfluence on Smooth Muscle
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20 . W .T . GERTHOFFER, J .S . FEDAN, D .P . WESTFALL, W .W . FLEMING and R .E . STITZEL, Proc . Fed . Amer . Soc . Ex~ . Biol . 36 1021 (1977) . 21 . 47.T . GERTHO~~, .S J . FEDAN, D .P . WESTFALL and W .W . FLEMING, Pharmacolo gist 19 243 (1977) . 22 . D .P . WESTFALL, J . Pharmacol . Exp . Ther . 20 1 267-275 (1977) . 23 . P .M . HUDGINS ans THARRfS, .M . J . Pharmac~o~. Ex~. Ther . 175 609-618 (1970) . 24 . 0 . CARRIER, Proc . Fed . Amer . Sôc . E x~. Biol . 34 11980 (1975) . 25 . P .W . ABEL, P .R . URQÛILLA and W .W . FLEMING, Proc. Fed . Amer . Soc . E~. Biol . 36 1028 (1977) . 26 . 0 . APRIGLIANO and K . HERMSMEYER, Circ . Res . 41 198-206 (1977) . 27 . D .A . TAYLOR, D .P . WESTFALL, S, de MORAÉS andW.W . FLEMING, Naunyn Schmiedeberg's Arch . Pharmacol . 293 81-87 (1976) . 28 . K . GOTO, D .P . WESTFALL and W .W . FLEMING, Pharmacologist 18 180 (1976) .
Pergamon Preee
THE TROPHIC INFLUENCE OF AUTONOMIC NERVES ON ELECTRICAL PROPERTIES OF THE CELL MEMBRANE IN SMOOTH MUSCLE William W . Flaming Department of Pharmacology West Virginia University Medical Center Morgantown, West Virginia
26506
Summary The relationship of neurotransmission to sensitivity in a variety of effector cell types is briefly surveyed . The several cellular changes which contribute to supersensitivity in dener vated skeletal muscle are listed . Subsequently evidence is reviewed regarding the role of a partial depolarization of the smooth muscle cells in the development of supersensitivity in the guinea-pig vas deferens . The depolarization appears to be the result of the decreased activity of an electrogenic Na+, K+ pump . The applicability of these findings to other autonomically innervated effectors is discussed . An intriguing relationship which is frequently manifest in research in the neurnsciences is the trophic relationship between nerves and effector cells . Not only do nerves demonstrate obvious moment-to-nroment regulation of effector cell activity, but they also have a long-term influence over many physiological and biochemical characteristics of the effector cells . Pharmacologic agents have provided a rich source of tools with which to study trophic relationships and, hence, pharmacologists have had a particular interest in these relationships . Our own laboratory has had a primary orientation toward the neurotrophic regulation of smooth muscle as manifest by enhanced sensitivity of the muscle to agonists consequent to chronic depression of neurotransmission . The phenomenon of denervation supersensitivity has been recognized for over one hundred years and has been the subject of considerable research (1) . However, the cellular mechanisms responsible for supersensitivity have been elusive, the more so because of the tendency of investigators to seek a single mechanism to explain the phenomenon . The first mechanism to be clearly identified occurs in skeletal muscle and was recognized through experiments which "mapped" the cholinoceptors on single muscle fibers by means of microelectrodes and microiontophoresis (2) . Such experiments led to the hypothesis that denervation supersensitivity in skeletal muscle is the result of a "spread" of receptors outward from the endplate . The validity of this hypothesis has subsequently become firmly supported by studies with the highly specific cholinoceptor-binding substance, 0300-9653/79/0410-1223$02 .00/0
Çopyright © 1978 Pergamon Press
1224
Neurotrophic Influence on Smooth Muscle
Vol . 22, No .e 13-15, 1978
a-bungerotoxin (3) . It is important to emphasize, however, that recent evidence clearly indicates that several different cellular mechanisms contribute to supersensitivity in skeletal muscle . These include receptor spread, complex electrophysiologic changes, altered calcium binding and a decrease in cholinesterase activity (4,5) . Interest in supersensitivity has gained renewed momentum in recent years with the recognition that denervation supersensitivity is an expression of a cellular homeostasis common to many types of excitable cells . The cells have the capacity to accomodate to chronic changes in the stimulus they receive and/or their own level of activity (6) . Excitable tissues in which supersensitivity has been demonstrated, include skeletal muscle, smooth muscle, cardiac muscle, exocrine glands, central neurons and the pineal gland . Given the evidence that multiple mechanisms contribute to supersensitivity in skeletal muscle and the physiologic diversity of the cells which can become supersensitive, it is obviously risky to assume that all of the same mechanisms contribute to supersensitivity in all types of cells . A major step in identifying the mechanisms responsible for supersensitivity in smooth muscle occurred when Trendelenburg (7) established that autonomic denervation produced two entirely separable types of supersensitivity . One type is consequent to inhibition or degeneration of a site of loss of a drug, such that a larger portion of the administered drug reaches the site of action . The sensitivity of the responding cells is not changed, only the distribution and/or fate of the agonist in the tissue . Examples are loss of adrenergic neuronal uptake (8) or extraneuronal uptake (9) . The other type of supersensitivity has been termed disuse, postsynaptic, postjunctional, or nondeviation supersensitivity (10) and represents an actual change in the sensitivity of the responding cells . It is this second type of supersensitivity which is under investigation in our laboratory and is the subject of this review . Postjunctional or nondeviation supersensitivity develops in many types of excitable cells whenever normal neuroeffector transmission is chronically and markedly depressed . The result is independent of the means used to inter rupt transmission (6) . In smooth muscle the supersensitivity is characterized by a delayed onset and nonspecificity, that is, the sensitivity is increased to a similar degree to several unrelated agonists . For example, in the guineapig vas deferens, depression of adrenergic transmission can be achieved by postganglionic denervation ("denervation"), preganglionic denervation ("decentralization") or chronic administration of reserpine (11,12) . Supersensitivity appears and is fully developed four days after impairment of transmission . The dose-response curves for norepinephrine, acetylcholine, histamine and potassium are all shifted to the left by a mean factor of 2 to 4 (12) . The nonspecificity of supersensitivity in smooth muscle led to the conclusion that neither a qualitative nor a quantitative change in receptors is likely to be the primary mechanism for the sensitivity change (13) . Subse quently, indirect evidence led to the hypothesis that a partial depolarization contributes to supersensitivity in smooth muscle (14) . More recent work in our laboratory has been aimed at directly measuring electrophysiologic and biochemical changes in supersensitive smooth muscle, particularly that of the guinea-pig vas deferens . Intracellular recording established that the membrane potential is reduced by 8-10 mV by chronic denervation or decentralization of the vas deferens . The time-course of the depolarization is the same as the time-course of the appearance of the supersensitivity (15) . Of equal significance, the threshold level at which action
Vol . 22, No .s 13-15, 1978
Neurotrophic Influence on Smooth Muscle
1225
potentials are elicited is unchanged (16) . Thus, the average amount of depolarization necessary to induce contraction is reduced by 35% by denervation . Consequently, a smaller concentration of a depolarizing agonist would be required to produce contraction . If, as the above results suggest, a partial depolarization contributes to the increased sensitivity of the smooth muscle cells, any procedure which causes an acute depolarization of control cells should also render them supersensitive . There are several means by which the smooth muscle cells of the guinea-pig vas deferens may be depolarized, some of them dependent upon the fact that, in this organ, the Na+, K+ pump of the smooth muscle cells is electrogenic . If a Na+ , K+ pump is electrogenic, it transports ions unequally across the membrane, thus creating a current and directly contributing to the membrane potential . According to Droogmans and Casteels (17), the total membrane potential of a cell with an electrogenic pump has two components as follows : in which Em E d i tôtal membrâne potential, Ediff = diffusion potential, Rm = membrane resistance and i p = the electrogenic current . Ouabain, a specific inhibitor of the Na+, K+ pump, reduces Em by reducing ip . ChanQing the concentration of external potassium ([K+] ) can affect either Ediff lbY changing the concentration radient across tRe membrane) or i (by inhibiting or stimulating the Na , K+ pump) . The relationship of [~+] o to these factors in the guinea-pig vas deferens is such that either reducing the [K+] o from 5 .8 mM (control solution) to 2 .9 mM or increasing it from 5 .8 to 11 .6 mM causes a partial depolarization of the membrane . The effects of ouabain, low and high [K+] o on resting potential were tested with microelectrodes in paired vasa deferentia . Other pairs of vasa deferentia were used to determine the effects on dose-response curves of histamine and methoxamine (chosen instead of norepinephrine because its doseresponse curve is unaffected by alterations in adrenergic neuronal uptake) . Ouabain (10 - 5M), half normal [K+] o and twice normal [K+] o each caused a depolarization of 8-10 mV and increases of 2 .5- to 5-fold in sensitivity to histamine and methoxamine (18,19) . These results compare extremely well with decentralization and denervation which cause a depolarization of 8-10 mV and increases in sensitivity of 2- to 4- fold . It is concluded that the depolarization produced by denervation and decentralization is adequate to produce the supersensitivity which accompanies it . The amount of ouabain used in the above experiments, 10'SM, is approximately optimal for the inhibition of Na+, K+ ATPase in the vas deferens (unpublished observations) . The fact that it produces a depolarization similar in magnitude to that produced by decentralization and denervation suggests the possibility that the latter procedures may also inactivate the electrogenic component of the Na+, K+ pump . Support for this suggestion comes from the fact that ouabain has no effect whatsoever on the membrane potential or the sensitivity of chronically denervated vasa deferentia (unpublished observations) . Experiments were therefore carried out to determine if there were any differences between control and supersensitive guinea-pig vasa deferentia in the activity of ouabain-sensitive Na+, K+ ATPase . Chronic denervation, decentralization or reserpine pretreatment decreased the activity of the enzyme 25-50% (20,21 and unpublished observations) . Kinetic analyses have established that the decreased activity is associated with a decreased Vmax ; Kg is unchanged (unpublished observations) .
1226
Neurotrophic Influence on Smooth Muscla
Vol . 22, N6 .s 13-15, 1978
The evidence outlined above leads to the conclûsion that a partial depolarization of the smooth muscle membrane is an important contributing factor to supersensitivity in the guinea-pig vas deferens . It should be noted, how ever, that there is at least one other contributing factor . Westfall (22) has shown that there is still a difference in sensitivity to agonists between control and denervated vasa deferentia when they are completely depolarized . Westfall suggested that this second factor involves an alteration in calcium mobilization . There is evidence in vascular smooth muscle that calcium does playa role in supersensitivity (23,24) . An important question arises . Is the guinea-pig vas deferens typical of other smooth muscles in regard to the electrophysiologic contribution to supersensitivity? No other smooth muscle has been studied as thoroughly in this regard . However, there is some evidence for a role of membrane potential in supersensitivity in the guinea-pig ileum (14), the rabbit saphenous artery (25), the rat portal vein (26) and even in cardiac muscle (27) . The role of an electrogenic pump has not been investigated as yet in any of these except the saphenous artery . Our preliminary evidence in that organ is that an electrogenic pump may be involved (unpublished observations) . An interesting contrast is the rat vas deferens . The smooth muscle of that organ does not appear to have an electrogenic pump and does not undergo partial depolarization in association with denervation supersensitivity (28) . Instead, there is a change in the threshold for electrical activation . Thus, even in the rat vas deferens, membrane electrical properties do seem to contribute to supersensitivity . References 1 . W .B . CANNON and A . ROSENBLUETH, The Su ersensitivit of Denervated Structures, Macmillan, New York 949 . 2 . S . THE LE F, Physiol . Rev . 40 734-152 (1960) . 3 . S . THESLEFF, Ann . N .Y .~Icad.Sci . 228 89-103 (1974) . 4 . W .W . FLEMING,Rev . Neurost . X43-x(1976) . 5 . M .G . McCONNELLând L .L . SIMPSON, _J . Pharmacol . E~. Ther . 198 507-517 (1916) . 6 . W .W . FLEMING, J .J . McPHILLIPS and D .P . WESTFALL, Rev . P siol . _68 55-119 (1973) . 7 . U . TRENDELENBURG, Pharmacol . Rev . 15 225-276 (1963) . 8 . U . TRENDELENBURG, a~ rm~. Rév . T$ 629-640 (1966) . 9 . U . TRENDELENBURG and K .-H . GRAÉFE, Proc . _Fed . Amer . Soc . Ex~. Biol . _34 1971-1974 (1975) . 10 . W .W . FLEMING, Proc . Fed . Amer . Soc . ~Ex . Biol . 34 1969-1970 (1975) . Pharmacy 11 . D .P . WESTFALL, Brit . 39 110-1 (~0) . 12 . D .P . WESTFALL, D .C . McCLURE and W .W.FLEMING, J . Pharmacol . E~. Ther . 181 328-338 (1972) .. 13 . h. HUDGINS and W .W . FLEMING, J . Pharmacol . E~. Ther . 153 70-80 (1966) . 14 . W .W . FLEMING, J . Pharmacol . Exp. Ther . 1277 - 85 1968 15 . W .W . FLEMING and D .P . WESTFALL, JP~armâcol . E~. Ther . 192 381-389 . (1975) . 16 . K . GOTO, D .P . WESTFALL and W .W . FLEMING, _J . Pharmacol . E~ . Ther . (in press) . 17 . G . DROOGMANS and R . CASTEELS, P siolo of Smooth Muscle, p . 11, ed . E . Bülbring and M .F . Shuba, Raven ress, New Yorc 9 18 . P .R . URQUILLA and W .W . FLEMING, Pharmacol ogist 16 273 (1974) . 19 . P .R . URQUILLA and W .W . FLEMING, Proc . Fee ~. Amer . Soc . Ex~. Biol . 34 818 (1975) .
Vol . 22, No .s 13-15, 1978
Neurotrophic Tnfluence on Smooth Muscle
1227
20 . W .T . GERTHOFFER, J .S . FEDAN, D .P . WESTFALL, W .W . FLEMING and R .E . STITZEL, Proc . Fed . Amer . Soc . Ex~ . Biol . 36 1021 (1977) . 21 . 47.T . GERTHO~~, .S J . FEDAN, D .P . WESTFALL and W .W . FLEMING, Pharmacolo gist 19 243 (1977) . 22 . D .P . WESTFALL, J . Pharmacol . Exp . Ther . 20 1 267-275 (1977) . 23 . P .M . HUDGINS ans THARRfS, .M . J . Pharmac~o~. Ex~. Ther . 175 609-618 (1970) . 24 . 0 . CARRIER, Proc . Fed . Amer . Sôc . E x~. Biol . 34 11980 (1975) . 25 . P .W . ABEL, P .R . URQÛILLA and W .W . FLEMING, Proc. Fed . Amer . Soc . E~. Biol . 36 1028 (1977) . 26 . 0 . APRIGLIANO and K . HERMSMEYER, Circ . Res . 41 198-206 (1977) . 27 . D .A . TAYLOR, D .P . WESTFALL, S, de MORAÉS andW.W . FLEMING, Naunyn Schmiedeberg's Arch . Pharmacol . 293 81-87 (1976) . 28 . K . GOTO, D .P . WESTFALL and W .W . FLEMING, Pharmacologist 18 180 (1976) .
