Effects of isoproterenol constriction in vascular STEPHEN
F. FLAIM
AND
on adrenergic smooth muscle
ROBERT
ZELIS
Division of Cardiology, Department of Medicine and The Milton S. Hershey Medical Center, Pennsylvania School of Medicine, Hershey, Pennsylvania 17033
FLAW, STEPHEN F., AND ROBERT ZELIS. Effects ofisoprotereno2 on adrenergic constriction in llascular smooth muscle. Am. J. Physiol. 233(l): H22-H26, 1977 or Am. J. Physiol.: Heart Circ. Physiol. 2(l): H22-H26, 1977. -A series of isolated segments of carotid arteries from Dutch-belted, adult, male rabbits were studied with respect to their response to a beta receptor agonist. Segments of 3-cm length were mounted in a chamber with constant surrounding temperature and pressure and perfused at constant pressure. Inflow pressure, outflow pressure, and flow rate were measured, and values of resistance (R) were calculated. Subsequent to control R, each vessel was exposed to a vasoconstricting concentration of either norepinephrine (NE: 10pH, 10 g, 10-“) Mj or potassium (K’: 100 mM, 50 mM) f o 11owed by three doses of the p-adrenergic agonist, isoproterenol (IP: lo-+, lo-", lO+M) administered simultaneously with each constrictor. R was not altered by IP during NE infusion but was significantly increased at all levels of IP during both K+ infusions. When the K+ series was repeated with alpha blockade, IP did not alter R. Thus, beta receptors do not appear to be functionally present in the adult rabbit carotid artery. beta-adrenergic receptors; alpha-adrenergic lar resistance; norepinephrine; potassium; bit; isolated-perfused carotid artery
receptors; vascuphentolamine; rab-
Department of Physiology, State University
muscle. These studies have demonstrated beta receptor activity in rabbit (4, 6, 14, 18, Zl), guinea pig, and rat (4, 6). Fleisch and co-workers (6) found that not only were beta receptor activities more pronounced in the thoracic than in the abdominal aorta of rat, guinea pig, and rabbit, but that the intensity of the vascular relaxation response mediated by beta receptors is diminished in these species with age. Beta receptor responses have also been elicited in isolated segments of canine femoral artery (7). Herlihy and Murphy (9) were unable to show an active relaxation response in hog carotid artery with epinephrine and alpha receptor blockade, suggesting a functional absence of the beta receptor in this tissue. Thus, while the location of beta-adrenergic receptors throughout the regional circulation appears to be fairly well understood, the data describing the distribution of these receptors in large arteries are incomplete. Therefore, in order to test the uniformity of distribution of beta-adrenergic receptors in large arteries, a series of in vitro studies were conducted on isolated, intact segments of carotid artery taken from adult rabbits. METHODS
RECEPTORS are preSeTIt in VaSCUhr smooth muscle in various regions of the peripheral circulation. Following alpha-adrenergic receptor blockade, a vasodilator response to sympathetic nerve stimulation and/or to catecholamine injection has been demonstrated in the skeletal muscle (2, 201, cutaneous (191, mesenteric (16), intestinal (8), and coronary (14) vasculature while functional beta-adrenergic receptors appear to be almost totally absent in the cerebral circulation (22). Though the contribution of the vascular betaadrenergic receptor to the overall regulation of blood flow probably has its greatest significance at the level of small arteries and resistance vessels, a number of studies have successfully demonstrated the functional presence of these receptors in large vessels of various species. Both Aviado (1) and Somlyo and Woo (17) observed a beta-adrenergic vascular relaxation response to norepinephrine after alpha receptor blockade in isolated main pulmonary artery. The majority of studies on large vessel beta receptors, however, have utilized aortic smooth BETA-ADRENERGIC
The experimental techniques utilized in these studies have been described previously (3). A total of 25 carotid artery segments from 15 adult, male, Dutch-belted rabbits with an average body weight of 2.24 2 0.58 kg were used. Animals were killed by cervical fracture and 3-cm segments of the carotid arteries immediately excised. The excised segments were placed in room temperature Krebs solution, and the loose collagenous tissue sheath was removed. A segment was then transferred to a Lucite chamber and connected to inflow and outflow pipettes. The vessel was returned to its in situ length as previously described (3). The chamber was sealed and filled with Krebs solution at 37°C and aerated with gas containing 95% 0, and 5% CO,. The perfusion system is schematically represented in Fig. 1. Inflow pressure (P,), outflow pressure (P,,), and outflow rate (Q) were recorded on separate channels of a Beckman type R Dynograph recorder. Resistance to flow (R) was computed using the equation R = P, - I’,,/ Q. A fourth channel recorded surrounding pressure (P,) within the chamber which was maintained at 0 mmHg. Pressures were measured with Statham P23BB trans-
H22 Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 20, 2019.
ISOPROTERENOL
AND
VASCULAR
SMOOTH
HZ3
MUSCLE
presented as means t standard error of the mean. Statistical significance was determined using the Student ttest for paired data or for grouped means where appropriate. In four separate experiments, IP was tested on blood vessels previously constricted with the nonadrenergic agent 5=hydroxytryptamine (5, 23). In two of these studies, vasodilation was induced by the nonadrenergic vasodilator, NG. The ability of NG to induce vasodilation of the preparation during NE stimulation was also tested. RESULTS
FIG. 1. Schematic illustration of isolated, perfused rabbit carotid artery preparation: 1) carotid segment, 2) intraluminal perfusion reservoir, 3) inflow pressure (Pl>, 4) outflow pressure (P,,), 5) perfusion flow rate (&), 6) superfusion pressure (PSI, 7) recording system, 8) extraluminal superfusion reservoir, 9) aeration system, 10) superfusion temperature.
ducers and flow was recorded by a rotameter type flowmeter. The perfusion fluid was a modified Krebs solution. The adjustments in the. chemical composition which have been described (3) were designed to induce an osmotic pressure equal to that of blood plasma. The experimental solutions consisted of the perfusion fluid with the desired concentration of one or a combination of the following vasoactive agents: K+ (KCl), norepinephrine (NE) (Levophed bitartrate, Winthrop), isoproterenol (IP) (Isuprel hydrochloride, Winthrop), phentolamine (Phen) (Regitine mesylate, CIBA), 5=hydroxy= tryptamine (5=HT) (Sigma), and nitroglycerin (NG) (Nitrostat, Parke-Davis). Perfusion solutions were prepared immediately before use and sealed in lightopaque reservoirs to prevent oxidation of the adrenergic agents in Krebs solution. As a further retardant to oxidation, EDTA (ethylenediamine tetraacetic acid) was added to the Krebs solution to bind heavy metal ions (iron and cobalt) which enhance the oxidation rate of catecholamines. After a 2-h equilibration period the vessel was perfused with a control solution for approximately 3 min and a base-line resistance was computed. To ensure a stabilized response, the vessel was stimulated with a priming dose of 10wsM NE for a period of 3 min or until a steady-state resistance was recorded. The vessel was then perfused with the control solution until the original base line was again achieved. Priming procedures were repeated approximately three times for each vessel. The vessels were then subjected to one of several protocols designed to illustrate the effect of IP? at three concentration levels, on the constrictor response to either NE or K+. After determining the alpha-adrenergic receptor blocking concentration of Phen (2 pg/ml) during stimulation with NE ( lODaM), the K+ and IP studies were repeated in the presence of Phen. Data are
Response to IP during NE stimulation. Thirty-eight independent experiments were conducted on 11 carotid artery segments. Segments were tested at three concentrations of IP (1Uw7, IO+, 10s5M) during stimulation with NE at three steady-state concentrations (lO+$ The results are shown in Fig. 2. The lo+, lO+M), response of the vessel to each NE and IP combination was compared to the respective control response to NE alone at the corresponding steady-state concentration. During high-level vasoconstriction with NE (lo+ M), IP did not significantly alter vascular resistance. During moderate vasoconstriction with NE (lOvg M), IP did not affect vascular resistance except at its highest concentration where a small but significant increase in resistance was noted. During low-level vasoconstriction with NE (lo-l0 M) vascular resistance was unaltered by IP except at its’lowest concentration where a small but significant decrease in resistance was noted. Response to IP during K+ stimulation. Eighteen independent experiments were conducted on seven carotid artery segments. Basal tone was induced by steadystate K+ stimulation at two concentration levels (50 mM, 100 mM>. The response of the vessels to IP (10B7, lo-“, low5 M) during K+ stimulation was compared to the corresponding control response to K+ alone. The results are shown in Fig. 3. During low-level K+ vasoconstriction, IP significantly increased resistance at all IP concentrations. During high-level K+ vasoconstriction, IP I
I 0
I
I 10-7
i
I
T 1
3
I 1o-6
I 10-5
I
-
MOLARISOPROTERENOL 2. Response to isoproterenol during norepinephrine-induced steady-state vasoconstriction at two concentration levels (mean _+ SEm). Asterisk indicates statistically significant difference from control at the 5% level or greater. NE = norepinephrine. FIG.
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 20, 2019.
S. F. FLAIM
H24 I w
-
1 0
I
Kt= 100mM Kt=50mM
I
10-7
I
I
I
I
1e
I
NE= 10-8M -NE= 10-gM
T
T
0
I
l@
0.2
,ugm/ml
MOLAR ISOPROTERENOL
3. Response to isoproterenol during potassium-induced steady-state vasoconstriction at two concentration levels (mean t SEm). Asterisk indicates statistically significant difference from control. K+ = potassium. FIG.
2.0
PHENTOLAMINE
Change in response to norepinephrine during alpha receptor blockade with increasing concentrations of phentolamine (mean + SEm). NE = norepinephrine. FIG.
4.
I
I
again significantly trations.
ZELZS
I
I
0.8 ~
AND ROBERT
1
increased resistance at all IP concen-
Response to NE with Phen. To determine a concentration of Phen inducing near maximal alpha-adrenergic receptor blockade, 31 independent experiments were conducted on three carotid artery segments. The results are shown in Fig. 4. After determination of the unstimulated resistance value, segments were perfused with one of six experimental solutions in a random fashion. Solutions contained either the high NE level (10B8M) or the moderate NE level (lO+ M) alone or in combination with Phen at one of two concentration levels (0.2 or 2.0 ,ug/ml). The change in resistance from the control unstimulated state was measured for each solution. The responses to the NE-Phen solutions were compared with those to the NE solutions. Figure 4 shows that almost no change in resistance occurred during NE infusion at either concentration in the presence of Phen at 2 pg/ml. The 95% confidence intervals for the means at these points indicated that these responses were not significantly different from 0. Therefore, Phen at 2 pg/ml was chosen as an effective concentration for complete alphaadrenergic blockade. K+ and IP zuith and without Phen. The response of vessels to K+ and IP, as previously described, was repeated in the presence of Phen at 2 pg/ml. The change in resistance to each concentration of IF in the presence of K+ and Phen was compared to the control response to K+ and Phen alone. Figure 5 illustrates these data compared to the previous data without Phen which have been replotted. Statistical comparisons were made between data from with-Phen and no-Phen groups corresponding to the same concentration of K+ and IP, with respect to both the absolute response and to the percent response. At both K+ levels, IP induced increased vasoconstrictor responses. When the study was repeated with alpha-adrenergic receptor blockade, IP did nut induce substantial changes in the response. The withPhen responses were reduced as compared to the noPhen responses at all comparison points. The reductions were statistically significant at 11 of the 12 comparison points. Response to IP during 5-HT stimulation. The response of unprimed vessels to IP during submaximal vasoconstriction with 5-HT was measured. In several
o.q-
no Phen
-1
10
~~I
__-
_._--
-
-~
-s
W-P-I T----
---P --d
p
F. FLAIM
AND
on adrenergic smooth muscle
ROBERT
ZELIS
Division of Cardiology, Department of Medicine and The Milton S. Hershey Medical Center, Pennsylvania School of Medicine, Hershey, Pennsylvania 17033
FLAW, STEPHEN F., AND ROBERT ZELIS. Effects ofisoprotereno2 on adrenergic constriction in llascular smooth muscle. Am. J. Physiol. 233(l): H22-H26, 1977 or Am. J. Physiol.: Heart Circ. Physiol. 2(l): H22-H26, 1977. -A series of isolated segments of carotid arteries from Dutch-belted, adult, male rabbits were studied with respect to their response to a beta receptor agonist. Segments of 3-cm length were mounted in a chamber with constant surrounding temperature and pressure and perfused at constant pressure. Inflow pressure, outflow pressure, and flow rate were measured, and values of resistance (R) were calculated. Subsequent to control R, each vessel was exposed to a vasoconstricting concentration of either norepinephrine (NE: 10pH, 10 g, 10-“) Mj or potassium (K’: 100 mM, 50 mM) f o 11owed by three doses of the p-adrenergic agonist, isoproterenol (IP: lo-+, lo-", lO+M) administered simultaneously with each constrictor. R was not altered by IP during NE infusion but was significantly increased at all levels of IP during both K+ infusions. When the K+ series was repeated with alpha blockade, IP did not alter R. Thus, beta receptors do not appear to be functionally present in the adult rabbit carotid artery. beta-adrenergic receptors; alpha-adrenergic lar resistance; norepinephrine; potassium; bit; isolated-perfused carotid artery
receptors; vascuphentolamine; rab-
Department of Physiology, State University
muscle. These studies have demonstrated beta receptor activity in rabbit (4, 6, 14, 18, Zl), guinea pig, and rat (4, 6). Fleisch and co-workers (6) found that not only were beta receptor activities more pronounced in the thoracic than in the abdominal aorta of rat, guinea pig, and rabbit, but that the intensity of the vascular relaxation response mediated by beta receptors is diminished in these species with age. Beta receptor responses have also been elicited in isolated segments of canine femoral artery (7). Herlihy and Murphy (9) were unable to show an active relaxation response in hog carotid artery with epinephrine and alpha receptor blockade, suggesting a functional absence of the beta receptor in this tissue. Thus, while the location of beta-adrenergic receptors throughout the regional circulation appears to be fairly well understood, the data describing the distribution of these receptors in large arteries are incomplete. Therefore, in order to test the uniformity of distribution of beta-adrenergic receptors in large arteries, a series of in vitro studies were conducted on isolated, intact segments of carotid artery taken from adult rabbits. METHODS
RECEPTORS are preSeTIt in VaSCUhr smooth muscle in various regions of the peripheral circulation. Following alpha-adrenergic receptor blockade, a vasodilator response to sympathetic nerve stimulation and/or to catecholamine injection has been demonstrated in the skeletal muscle (2, 201, cutaneous (191, mesenteric (16), intestinal (8), and coronary (14) vasculature while functional beta-adrenergic receptors appear to be almost totally absent in the cerebral circulation (22). Though the contribution of the vascular betaadrenergic receptor to the overall regulation of blood flow probably has its greatest significance at the level of small arteries and resistance vessels, a number of studies have successfully demonstrated the functional presence of these receptors in large vessels of various species. Both Aviado (1) and Somlyo and Woo (17) observed a beta-adrenergic vascular relaxation response to norepinephrine after alpha receptor blockade in isolated main pulmonary artery. The majority of studies on large vessel beta receptors, however, have utilized aortic smooth BETA-ADRENERGIC
The experimental techniques utilized in these studies have been described previously (3). A total of 25 carotid artery segments from 15 adult, male, Dutch-belted rabbits with an average body weight of 2.24 2 0.58 kg were used. Animals were killed by cervical fracture and 3-cm segments of the carotid arteries immediately excised. The excised segments were placed in room temperature Krebs solution, and the loose collagenous tissue sheath was removed. A segment was then transferred to a Lucite chamber and connected to inflow and outflow pipettes. The vessel was returned to its in situ length as previously described (3). The chamber was sealed and filled with Krebs solution at 37°C and aerated with gas containing 95% 0, and 5% CO,. The perfusion system is schematically represented in Fig. 1. Inflow pressure (P,), outflow pressure (P,,), and outflow rate (Q) were recorded on separate channels of a Beckman type R Dynograph recorder. Resistance to flow (R) was computed using the equation R = P, - I’,,/ Q. A fourth channel recorded surrounding pressure (P,) within the chamber which was maintained at 0 mmHg. Pressures were measured with Statham P23BB trans-
H22 Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 20, 2019.
ISOPROTERENOL
AND
VASCULAR
SMOOTH
HZ3
MUSCLE
presented as means t standard error of the mean. Statistical significance was determined using the Student ttest for paired data or for grouped means where appropriate. In four separate experiments, IP was tested on blood vessels previously constricted with the nonadrenergic agent 5=hydroxytryptamine (5, 23). In two of these studies, vasodilation was induced by the nonadrenergic vasodilator, NG. The ability of NG to induce vasodilation of the preparation during NE stimulation was also tested. RESULTS
FIG. 1. Schematic illustration of isolated, perfused rabbit carotid artery preparation: 1) carotid segment, 2) intraluminal perfusion reservoir, 3) inflow pressure (Pl>, 4) outflow pressure (P,,), 5) perfusion flow rate (&), 6) superfusion pressure (PSI, 7) recording system, 8) extraluminal superfusion reservoir, 9) aeration system, 10) superfusion temperature.
ducers and flow was recorded by a rotameter type flowmeter. The perfusion fluid was a modified Krebs solution. The adjustments in the. chemical composition which have been described (3) were designed to induce an osmotic pressure equal to that of blood plasma. The experimental solutions consisted of the perfusion fluid with the desired concentration of one or a combination of the following vasoactive agents: K+ (KCl), norepinephrine (NE) (Levophed bitartrate, Winthrop), isoproterenol (IP) (Isuprel hydrochloride, Winthrop), phentolamine (Phen) (Regitine mesylate, CIBA), 5=hydroxy= tryptamine (5=HT) (Sigma), and nitroglycerin (NG) (Nitrostat, Parke-Davis). Perfusion solutions were prepared immediately before use and sealed in lightopaque reservoirs to prevent oxidation of the adrenergic agents in Krebs solution. As a further retardant to oxidation, EDTA (ethylenediamine tetraacetic acid) was added to the Krebs solution to bind heavy metal ions (iron and cobalt) which enhance the oxidation rate of catecholamines. After a 2-h equilibration period the vessel was perfused with a control solution for approximately 3 min and a base-line resistance was computed. To ensure a stabilized response, the vessel was stimulated with a priming dose of 10wsM NE for a period of 3 min or until a steady-state resistance was recorded. The vessel was then perfused with the control solution until the original base line was again achieved. Priming procedures were repeated approximately three times for each vessel. The vessels were then subjected to one of several protocols designed to illustrate the effect of IP? at three concentration levels, on the constrictor response to either NE or K+. After determining the alpha-adrenergic receptor blocking concentration of Phen (2 pg/ml) during stimulation with NE ( lODaM), the K+ and IP studies were repeated in the presence of Phen. Data are
Response to IP during NE stimulation. Thirty-eight independent experiments were conducted on 11 carotid artery segments. Segments were tested at three concentrations of IP (1Uw7, IO+, 10s5M) during stimulation with NE at three steady-state concentrations (lO+$ The results are shown in Fig. 2. The lo+, lO+M), response of the vessel to each NE and IP combination was compared to the respective control response to NE alone at the corresponding steady-state concentration. During high-level vasoconstriction with NE (lo+ M), IP did not significantly alter vascular resistance. During moderate vasoconstriction with NE (lOvg M), IP did not affect vascular resistance except at its highest concentration where a small but significant increase in resistance was noted. During low-level vasoconstriction with NE (lo-l0 M) vascular resistance was unaltered by IP except at its’lowest concentration where a small but significant decrease in resistance was noted. Response to IP during K+ stimulation. Eighteen independent experiments were conducted on seven carotid artery segments. Basal tone was induced by steadystate K+ stimulation at two concentration levels (50 mM, 100 mM>. The response of the vessels to IP (10B7, lo-“, low5 M) during K+ stimulation was compared to the corresponding control response to K+ alone. The results are shown in Fig. 3. During low-level K+ vasoconstriction, IP significantly increased resistance at all IP concentrations. During high-level K+ vasoconstriction, IP I
I 0
I
I 10-7
i
I
T 1
3
I 1o-6
I 10-5
I
-
MOLARISOPROTERENOL 2. Response to isoproterenol during norepinephrine-induced steady-state vasoconstriction at two concentration levels (mean _+ SEm). Asterisk indicates statistically significant difference from control at the 5% level or greater. NE = norepinephrine. FIG.
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 20, 2019.
S. F. FLAIM
H24 I w
-
1 0
I
Kt= 100mM Kt=50mM
I
10-7
I
I
I
I
1e
I
NE= 10-8M -NE= 10-gM
T
T
0
I
l@
0.2
,ugm/ml
MOLAR ISOPROTERENOL
3. Response to isoproterenol during potassium-induced steady-state vasoconstriction at two concentration levels (mean t SEm). Asterisk indicates statistically significant difference from control. K+ = potassium. FIG.
2.0
PHENTOLAMINE
Change in response to norepinephrine during alpha receptor blockade with increasing concentrations of phentolamine (mean + SEm). NE = norepinephrine. FIG.
4.
I
I
again significantly trations.
ZELZS
I
I
0.8 ~
AND ROBERT
1
increased resistance at all IP concen-
Response to NE with Phen. To determine a concentration of Phen inducing near maximal alpha-adrenergic receptor blockade, 31 independent experiments were conducted on three carotid artery segments. The results are shown in Fig. 4. After determination of the unstimulated resistance value, segments were perfused with one of six experimental solutions in a random fashion. Solutions contained either the high NE level (10B8M) or the moderate NE level (lO+ M) alone or in combination with Phen at one of two concentration levels (0.2 or 2.0 ,ug/ml). The change in resistance from the control unstimulated state was measured for each solution. The responses to the NE-Phen solutions were compared with those to the NE solutions. Figure 4 shows that almost no change in resistance occurred during NE infusion at either concentration in the presence of Phen at 2 pg/ml. The 95% confidence intervals for the means at these points indicated that these responses were not significantly different from 0. Therefore, Phen at 2 pg/ml was chosen as an effective concentration for complete alphaadrenergic blockade. K+ and IP zuith and without Phen. The response of vessels to K+ and IP, as previously described, was repeated in the presence of Phen at 2 pg/ml. The change in resistance to each concentration of IF in the presence of K+ and Phen was compared to the control response to K+ and Phen alone. Figure 5 illustrates these data compared to the previous data without Phen which have been replotted. Statistical comparisons were made between data from with-Phen and no-Phen groups corresponding to the same concentration of K+ and IP, with respect to both the absolute response and to the percent response. At both K+ levels, IP induced increased vasoconstrictor responses. When the study was repeated with alpha-adrenergic receptor blockade, IP did nut induce substantial changes in the response. The withPhen responses were reduced as compared to the noPhen responses at all comparison points. The reductions were statistically significant at 11 of the 12 comparison points. Response to IP during 5-HT stimulation. The response of unprimed vessels to IP during submaximal vasoconstriction with 5-HT was measured. In several
o.q-
no Phen
-1
10
~~I
__-
_._--
-
-~
-s
W-P-I T----
---P --d
p
