Indian J Physiol Pharmacol 1996; 40(2): 120-126

INTERACTION OF CALCIUM CHANNEL BLOCKERS (CCBS) WITHHISTAMINE AND 5-HYDROXYTRYPTAMINE IN AORTA FROMNORMAL AND DISEASED RATS

PRAVEEN BHUGRA* AND O. D. GULATP*

Department ofPharmacology,Pramukhswami Medical College,Karamsad - 388325

( Received on September 28,1995 )

Abstract: The present study attempts to investigate the interaction ofcalcium rhannel blockers (CCEs) with histamine (H) and5-hydroxytryptamine (5-HT) in rat isolated aortic strip preparations.In preparations obtained from rats chronically treated with variousCCEs the contractile responses to H were completely blocked suggestingthat this may be due to inhibition of the voltage-dependent channelsand inositol 1,4,5-triphosphate induced release of calcium fromintracellular stores. The decreased contractions of the aortic strippreparations with 5-HT obtained from rats chronically treated withvarious CeEs implies a decre<1se in 5-HT receptor density. DOCA­saline hypertensive rats chronically treated with various CCEs showedvariable responses to Hand G-HT suggesting that these changes maybe due to different isoforms of L-type calcium channels. In L-thyroxine­treated preparations or those simult<1neously treated with L-thyroxineand CCEs the responses to H were abolished and thosl~ to 5-HT werepartially blocked with decrease in mdxima which could be secondaryto the primary effect on the heart and to generalised reduced senstivityof the rat aorta.

Key words: aortahistamine

INTRODUCTION

The contractile process in vascular smoothmuscle is dependent upon the presence of freecalcium ion at sufficient intracellularconcentration 0-3). A generally accepted schemeto expl<:>i;l the respon 'es of blood vessels toagonists such as noradrenaline, angiotensin and5-hydroxytrytamine (5-HT) is as follows (4) :

i) Breakdown of phosphatidyl inositol (PI)to yield triphosphoinositol (IP) which releasescalcium from intracellular stores.

ii) Opening of receptor-operated calciumchannels (ROC) which allows calcium to enterthe cell and produce depolarization.

5-hydroxytryptam i necalcium channel blockers

iii) Depolarization causes potential-operatedchannels (POC) to open, which allows calciumto enter the cell.

alcium channel blockers are establishedas one of the first line drugs III the therapy ofessential hypertension (f)). Increases in the K

Dand B values for [,3HJ nitrendipine bindingsites l~'~~'e been observed in heart membranesfrom 24-weeks old spontaneously hypertensiverats (SHR) (6). Clinically hyperthyroidism maybe associated with systolic hypertension (7)

Chronic treatment of cultured chick ventricularcell with thyroid hormon is reported to producean increase in calcium channel density whichcorrelat'd well with l,4-dihydropyridine

*COiTespondinr; Author-"Present ilddress . Ambalal Sambhai Enterprises. Wadi Wadi, Baroda - 390 007

Indian J Physiol Pharmacol 1996; 40(2) Calcium Channel Blockers, Histamine and 5-HT on Rat Aorta 121

sensitive calcium channel uptake (8). Incontrast, heart membranes from rats madehyperthyroid by 5-day treatment with thyroxineshowed a decrease in the number of VDCCs (9).

The present study attempts to investigatethe interaction of calcium channel blockers withhistamine (H), and 5-hydroxytryptamine (5-HT)in aorta from normal rats, DOCA-salinehypertensive rats and hyperthyroid rats.

METHODS

I. Preparation of rat isolated aortic strip fOl'

recording of contractions:

Male albino rats of Wistar strain weighing250-350 gm were used for the presentexperiments. Chronic treatment of the rats wasstarted from day one and at the time of sacrificethe rats weighed between 200 to 350 gmdepending on the chronic treatment withdifferent drugs. The animals were sacrificed bya sharp blow on the head and cutting the neckblood vessels.

The thoracic aorta was rapidly removedand placed into a petridish containingoxygenated McEwen physiological salt solution(pH-7.3) of the following composition (mM) :NaCI-ll3; KCI-5.6; CaCI

2-2.2; NaHC0

3-25.0,

NaH2P04-1.21; glucose-ll.l; and sucrose-13.1.The thoracic aorta was cleaned of connectivetissue and adherent fat. The isolated arterywas cut helically into strips as described byFurchgott and Bhadrakom (0) for the rabbitaortic strip. From each thoracic aorta stripswere prepared and the same portion of thestrip was used in all sets of experiments for aparticular agonist response.

The aortic strip was suspended into 30 m1organ bath containing McEwen physiologicalsalt solution (pH-7.3) maintained at 30°C ±

OSC. The bath medium was bubbled withoxygen (ll). The aortic strip was arranged forisometric tension recording by tying the lowerend to the glass tissue holder and upper end tothe force displacement transducer. The tissuewas allowed to equilibrate under 1 g restingtension for 60 min, during which the bathing

solution was routinely changed every 15 min.After equilibration the strips were exposed tocumulative doses of histamine (H) and 5-RT.The doses were added in a cumulative fashionat 0.3 log unit intervals. The next higher dosewas added after the first dose had causedmaximal contraction.

II. Chronic drug treatment schedules:

Group of 5-10 normal or 3-4 hypertensiverats received similar chronic treatments withdrugs.

Verapamil (30 mglkg) and diltiazem (20 mg/kg) dissolved in triple glass distilled water andnifedipine (10 mg/kg) and nimodipine (20 mg/kg) dissolved in the solvent (PEG400-969 g,glycerine-60 g, water-lOO g) were administeredorally via a Ryles tube once daily for 28 days(12).

Drug treatment schedules for thyroxine­treated rats were as follows :

A group of rats received simultaneously thesame dose of L-thyroxine sodium and nifedipine(10 mglkg) or nimodipine (20 mglkg) orally byRyles tube.

III. Diseased state models:

1. Hypertension.' Male albino rats weighingabout 100 g were kept on a diet high in sodiumchloride and drinking water was replaced by 2%sodium chloride solution ad lib. When theyattained a weight of about 250 g, they were alsogiven deoxycorticosterone acetate (DOCA)dissolved in sesame seed oil in a dose of 10 mg/kg, sc twice weekly for 42 days (13).

In order to check as to whether hypertensionhad been produced by the DOCA-salinetreatment schedule, blood pressure of rats wasrecorded (14).

Following confirmation of induction ofhypertension groups of 3-4 rats received chronictreatment with drugs as under II.

2. Hyperthyroidism .' Hyperthyroidism wasinduced by subcutaneous injection of 0.75 mg/

122 Bhugra and Gulati

kg L-thyroxine sodium in alkaline salinesolution (O.OOIN NaOH in 0.9% NaCl) daily for7 days (15).

Rectal temperature and heart weights ofL-thyroxine treated animals were recordedbefore the start of the treatment and beforesacrifice. Serum was pI' pared from bloodsamples collected from the common carotidartery of rat exsanuguinated on the day of theexperiment and stored at -20°C. Total serumthyroxine (T

4), Tri-iodothronine (T) and

thyroxine stimulating hormone (TSH) levelswere determined by enzyme immunoassay (EIA)with a commercially available in vitro 'diagnostickit' (Bin Me'rieux, France) on semi-autoanalyser(SEAC CH-lOO; ames marketed by Miles India,Baroda).

IV. Drugs:

Histamine (H), 5-hydroxytryptamine creatines Ifate complex (S-HT) was obtained from SigmaChemical Co., Pentobarbitone sodium wasobtained from National Chemicals, Baroda. Thefollowing drugs were received as free gifts :diltiazem (Sun Pharmaceutical Industries,Baroda); verapamil and nifedipine (TorrentPharmaceutical Ltd., Ahmedabad);L-thyroxine sodium (Glaxo Laboratoreis (India)Ltd., Bombay); nimodipine (U.S. Vitamins,Bombay), deoxycorticosterone acetate Onfar(Inda) Ltd., Calcutta).

Polyethylene glycol 400 (E. Merck (India)Ltd., Bombay); glycerine J.P. (MetroPharmaceutical Industry, Wadhwan City);sesame seed oil (Ahmed Mills, Bombay) wereobtained and used as solvents of the drugs.

The in vitro diagnostic kits used for theestimation of TSH, T

3and T

4by enzyme

immunoassay were of Bio Me'rieux, Franceobtained from "Cadila Diagnostic Division",Ahmedabad.

V. Statistical analysis:

Only one agonist was used for gettingconcentration-response curve in a givenpreparation. Contractile force was expressed as

Indian J Physial Pharmacal 1996; 40(2)

mg of tension developed. EC50 values weredetermined by the method of Van Rossum (16)from the concentration-response curve againstpercentage of its own maximum produced by agiven agonist. The pD~ values were determinedby the method of Ariens (17). The results areexpressed as mean ± S.E.M. and analysed bythe Student's "t" test for obtaining thE' level ofsignificance (18).

RESULTS

H (0.59xlO,7M-6.l6xlO· 5M) and 5-HT(0.74x lO'7M-9.70xlO·6M) produced concentration­dependent contractions of the aortic strip. Theconcentration-response curves of Hand 5-HTwere not significantly (P>0.05) affected inpreparations obtained from rats treated withsolvent (PEG 400-969 g; glycerine-60 g, water100 g), saline or sesame seed oil.

Effect of calcium channel blockers on the contractile

responses to Hand 5-HT:

Control rats: In preparations obtained fromrats chronically treated with verapamil,nifedipine, nimodipine or diltiazem the maximalcontractions generated by aortic strips inresponse to 5-HT and the pD

2values of 5-RT

compared to controls were significantly reduced.There was complete inhibition of l' sponses to H(0.59 x lO'7M - 4.72 x 10·3M) with all the calciumchannel blockers except verapamil which causeda significant reduction in the contractile forceand pD

2value of H as compared to control

(Table D.

Hypertensive rats: The mean blood pressureof control rats given sesame seed oil sc for 42days was 103 ± 3.3 mm Hg (n=3); the meanblood pressure of rats treated with DOCA ­saline for 42 days was 132.5 ± 2.5 mm Hg (n=4).The rise in blood pressure was significantly(P<O.Ol) higher.

In hypertensive preparations the contractileforce generated by the aortic strip in responseto Hand 5-HT was reduced significantly inrelation to control preparation. However, thepD2 values of the agonists were not significantly

Indian J Physiol Pharmacol 1996; 40(2) Calcium Channel Blockers, Histamine and 5-HT on Rat Aorta 123

TABLE I: Effect of CCBs on maximal contractile force and pDz generated in rat isolated aorta by 5-HT and histamine incontrol, DOCA-saline hypertensive and hyperthyroid rats.

Treatment group Maximumcontractileforce (mg)

5-HT

(n) pD2

valueMaximumcontractileforce (mg)

Histamine

(n) pD2

value

ControlChronic VerapamilChronic NifedipineChronic NimodipineChronic DiltiazenDOCA - salineDOCA - saline+Chronic NifedipineDOCA - saline+Chronic NimodipineL-ThyroxineL-Thyroxine+Chronic NifedipineL-Thyroxine+Chronic Nimodpine

630 ± 25225 ± 19'214 ± 14'504 ± 15*184 ± 08*306 ± 10*306 ± 58'"271 ± IONS282 ± 06"255 ± 00'"210 ± 06*

flO) 5.99 ± 0.04 774 ± 22 (8)(6) 5.04 ± 0.03* 372 ± 09* (6)(5) 5.01 ± 0.06* No response (5)(5) 5.62 ± 0.03** No response (5)(5) 4.42 ± 0.05* No response (5)(3) 6.08 ± 0.07NS 252 ± 18* (3)(3) 5.77 ± 0.01** 198 ± 10* (3)(3) 6.07 ± O.OlNS 220 ± 22NS (3)(3) 6.18 ± 0.06"s 228 ± 26* (3)(3) 6.27 ± 0.04N8 No response (3)(3) 6.27 ± 0.04N5 No response (3)

5.27 ± 0.054.83 ± 0.04*

5.27 ± 0.01 NS5.54 ± 0.14N5

5.60 ± O.IONS5.60 ± 0.07*

Values arc cited as means ± S.E.M.*P<O.Ol and "P<0.05 indicate significant differences.

Comporisons were made between (i) chronic calcium channel blockeriDOCA-salinelL-thyroxine and control; (ii) chronic DOCA­saline and chronic DOCA-saline + calcium channel blocker, (iii) chronic L-thyroxine and L-thyroxine + calcium channel blocker.

affected. Chronic treatment with nifedipine ornimodipine did not produce any significantchanges in the contractile force in response to5-HT as compared to that in DOCA - salinecontrol preparations. However, the pDz value of5-HT was significantly reduced by nifedipine,but was unaffected by nimodipine. In case of H,the maximal contractile force was significantlyreduced by chronic nifedipine treatment withoutany change in the pDz value of H. Chronicnimodipine neither significantly affected themaximal contractile force nor the pDz value(Table 1).

Hyperthyroid rats : Various somaticparameters were measured following inductionof hyperthyroid state and treatment withcalcium channel blockers. This was done tomonitor the efficacy of various treatmentprotocols. In hyperthyroid state the heart weightwas increased (P<O.Ol), the rectal temperaturewas enhanced (P<O.Ol), the T

3and T

4levels was

raised (P<O.Ol) and the TSH level was reduced(P<O.Ol). Chronic nimodipine and nifedipinetreatments reduced (P<O.Ol) the heart weightand rectal temperature, had no effect (P>0.05)on T'j and T

4levels and enhanced (P<O.Ol) the

TSH levels (Table Il).

Chronic treatment with L-thyroxinesignificantly (P<O.Ol) reduced the maximalcontractile force generated with both 5-HT andH; however the senstivity was reduced (P<O.Ol)only in the case of H as compared to control.Chronic treatment with L-thyroxine andnifedipine or nimodipine significantly (P<0.05;P<O.Ol) reduced the contractile force with 5-HT,but had no significant (P>0.05) effect on the pDzvalues as compared to L-thyroxine control. Bothnifedipine and nimodipine treatments abolishedresponses to H (Table n.

DISCUSSION

H can initiate smooth muscle contractionby releasing calcium from intracellular storesor by opening either receptor-operated orvoltage-operated calcium channels (19). Calciumfrom intracellular stores released by H-receptorstimulation has been demonstrated inendothelial cells (20), air way smooth muscle(21), rat aorta (22), although in most casesthere is also an appreciable stimulation oftransmembrane calcium influx (23).

In the present study with aortic strippreparations treated with verapamil there mayhave been some decrease in the density of H

124 Bhugra and Gulati Indian J Physiol Pharmacol 1996; 40(2)

TABLE II: Effect of chronic treatment with calcium channel blockers on various somatic parameters measured followingaltered thyroid state in rats.

Treatment group

Variable Control L-thyroxine L-thyroxine +

nij'edipine

L-thyroxine +

nimodipine

Body weight (g)

Rectal temperature in 'cHeart weight (ng 100"g body wt.)

Serum thyroxine (T) (MOG%)

Serum triodothyronine (T) (NG/l\1L)

Serum TSH (Mu/l\1Ll

250.00 ± 0.0

37.81 ± 0.03

362.00 ± 7.0

11.30 ± 0.3

1.23 ± 0.08

3.83 ± 0.03

216.66 ± 10.54*

39.56 ± 0.15*

594.00 ± 1.8*

38.26 ± 0.35*

8.68 ± 0.5*

0.24 ± 0.02*

316.66 ± 10.15*

37.75 ± 0.14*

407.30 ± 14.8*

38.26 ± 0.35NS

8.87 ± 0.13NS

1.60 ± 0.33*

225.00 ± 11.18NS

37.75 ± 0.17*

552.50 ± 22.0*

36.46 ± 1.37NS

7.00 ± 1.90NS

2.10 ± 0.90NS

Values are cited as means ± S.E.M.*P<O.OI and **P<0.05 indicate significant differences.

Comparison were made betweem (i) chronic L-thyroxine treatment and control (ij) chronic L-thyroxine treatment and chronicL-thyroxine + calcium channel blocker treatment.

receptors as evidenced by decrease in the pDzvalue. This is similar to the finding of Morel eta1 (24) who have shown that the contractileresponse to HI receptor stimulation in theintestinal smooth muscle is largely sensitive toinhibition by dihydropyridine-based antagonistsof voltage-dependent calcium channels.

The tension developed at high concentrationsof H in intestinal smooth muscle is partlyresistant to nifedipine indicating that acomponent of the contractile response may bemediated by inositol 1,4,5-triphosphate-inducedrelease of calcium from intracellular stores(24, 25). In the present study with chronicalIynifedipine, diltiazem or nimodipine-treatedpreparations, there was complete block ofcontractile response to H suggesting that thismay have been mediated by inhibition of inositol1,4,5 triphosphate-induced release of calciumfrom intracellular stores.

5-HT is considered to contract the rat aortaby stimulating 5-HTz receptors only. Alpha­adrenoceptor are not involved as prazosin andidazoxan (selective alpha and alpha zadrenoceptor antagonists, respectively) have noeffect on the 5-HT responses (26). In the presentstudy chronic treatment with verapamil,nifedipine, diltiazem or nimodipine reducedcontractile force and senstivity of 5-HT receptordensity.

Chronic nifedipine treatment of DOCA-salinehypertensive rats reduced the contractile forcewith H, but had no significant effect onthe contractile force with 5-HT. This treatmentdid not affect the senstivity to both theagonists. It is possible that these differentialchanges with the two calcium channelblockers may be releated to the occurenceof different isoforms of L-type calciumchannels (27).

In L-thyroxine treated preparations thecontractile force with both 5-RT and H wasreduced but the senstivity was reduced onlyin the case of H. The pDz value of 5-HTwas unaffected, but the maximal responsewas decreased by chronic treatment withthyroxine and nifedipine or nimodipine.Simultaneous treatment with nifedipine ornimodipine and L-thyroxine abolished theconcentration response of H. These effectscould be due to the generalised reducedsenstivity of the rat aorta and or secondary toa primary effect exerted on the heart.A significant reduction in heart weight(Table II) produced by chronic nifedipine ornimodipine administration in the thyroxinetreated rats would support the latter suggestion.Further work with specific radioligandswould be necessary to confirm the abovesuggestion.

Indian J Physiol Pharmacol 1996; 40(2) Calcium Channel Blockers, Histamine and 5-HT on Rat Aorta 125

ACKNOWLEDGEMENTS

The work was supported by a grant from

the Charutar Arogya MandaI Medical ResearchSociety, Karamsad, Gujarat.

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