- Email: [email protected]
Transmembrane ionic transport systems and hypertension
Transmembrane Ionic Transport Systems and Hypertension
MICHEL LAZDUNSKI, D.Sc. Nice, France
Various systems contribute to the regulation of intracellular calcium. The most important are: (1) the voltage-dependent calcium channel; (2) sodium/calcium exchange, resulting in a correlation between intracellular sodium and calcium; (3) plasma membrane calcium ATPase; and (4) the inositol triphosphate-regulated channel, releasing calcium from the endoplasmic and sarcoplasmic reticulum stores. The essential properties of these different systems, their pharmacologic features, and their regulation by various agonists (beta- and alpha-adrenergic angiotensin-type peptides, etc.) must be considered both for an understanding of the pathophysiology of hypertension and for its treatment. The aim of this study was to examine the various ionic transport systems --essentially those involved in sodium and calcium transport--that are the basis for smooth muscle and cardiac muscle contraction, and to study the molecular pharmacologic aspects of these transport systems.
CALCIUM TRANSPORT An absolutely fundamental point is that in every cell--not just cardiac or vascular cells--the external calcium concentration is much greater than the internal calcium concentration (Figure 1). The external concentration of ionized calcium is approximately 1.8 mM, whereas the cytoplasmic concentration, during the period of relaxation of the muscle cells, is about 0.1 /~M. A number of calcium transport systems are necessary to maintain this calcium gradient between the exterior and the interior of the cell.
EXTERNAL MEMBRANE SYSTEMS
From the Centre de Biochimiedu Centre National de la Recherche Scientifique (CNRS), Nice, France. Requestsfor reprintsshould be addressed to Professor Michel Lazdunski, Centre de Biochimie du CNRS, Parc Valrose, 06034 Nice Cedex, France.
The systems represented in Figure 1 are calcium transport systems located in the plasma membrane of cardiac and smooth muscle cells. Efflux System. The major system of efflux of calcium from the interior of the cell towards the exterior is not specific to smooth muscle cells or to cardiac cells, but is present in all cells. Calcium ions are taken from the interior of the cell and are expelled towards the exterior. As this operation occurs in the presence of an ionic gradient, it requires the hydrolysis of ATP, which explains the necessary intervention of an enzyme known as calcium ATPase [1]. Influx Systems. There are two systems of calcium influx in the plasma membrane. The first is the calcium channel, which represents the major system of calcium influx in smooth muscle and cardiac muscle plasma membranes [2-4]. This calcium channel will be discussed in more detail in relation to calcium channel inhibitors. The other calcium influx system in the plasma membrane, in reality, can be used either as an influx sys-
January 29, 1988 The American Journal of Medicine Volume84 (suppl 1B)
3
SYMPOSIUMON INDAPAMIDE--LAZDUNSKI
Blockedby Ca2" channel blockers
C a ;t+
/ °-°;°
--,~
Ca 2"
÷ + + ++ - - ~ - __~ __~ _ _ ~ " - ) ~ _ _ ~ l ~
+ -
Oiiw eleCgrr°~l . cihaeenr~ lj'
1.8 mM
polarizedmembrane (normalstate} "
/
[Ca2"1,=0.1 pM
• depolarized membrane
\
, \oo .a@
CH1~cHICH~
CH3
,
1
H~CO---I~'I~---"l m C - - (CHT)~ - N -
II
H3C0R'~ ~
J
Nokoown
pharmacology
I
Ca
(CH;,)~.---l;~l~
OCH~
~
OCH~
I
C-- N
Verapamll .
T~7 •
(R--H); D600 : (R--0CH3)
2.
0Me
I I I
Weakly blocked by amiloridederivatives Figure 1. Systems regulating calcium transport in the plasma membrane of cardiac and vascular cells.
I
o
CH~-CHe-NMe e Diltlazem
tern or as an efflux system. This system of reversible ex, change, which links the efflux of sodium with the influx of calcium or vice versa, is called the sodium/calcium exchange system [5]. Pharmacologic Aspects. On the basis of this schematic representation, it is possible to imagine a variety of molecules that coutd be active in hypertension. A molecute could stimulate calcium ATPase, i.e., expel calcium from the cell in order to induce less contraction; however, at the present time, the pharmacology of calcium ATPase is unknown. A molecule could be an effector of the sodium/ calcium exchange system, but the pharmacology of this exchange has not been well defined (although it can be blocked by substitution of sodium with lithium and, to a lesser degree, by certain derivatives of amiloride). In contrast, the pharmacology of the calcium channels has been perfectly defined. Figure 2 summarizes a number of data concerning calcium channel inhibitors. At the top of Figure 2, a diagram explains the functioning of the plasma membrane calcium channel. This calcium channel is a protein that has now been purified and its structure clearly identified [6-8]. This protein pore can be either closed or open and therefore permeable to calcium, depending on the membrane potential. It opens during the action potential and allows influx of calcium, which subsequently induces contraction. The calcium channel is blocked by a whole series of inhibitors, the principal examples of which are presented in the center of Figure 2. These are molecules of the verapamil type, the diltiazem type, and the dihydropyridine type (ni-
4
January 29, 1988
The American Journal of Medicine
~N07
[ ~
1
NO~
I
H
H
Nlfedlplne
Nitrendiplne
DEPOLARIZED 1
Hy[~r~ pERpo E.=~O~=. LARIZ ..~=D Ko~= 13nM
E .5 Z Kos=0.15nM • 0 L . . , -12 -11 -10 -9 -8 -7 -6 -5 Log [ (+) PN200-- 110] Figure 2. Pharmacologic aspects ofthe calcium channels (reproduced from [12]).
fedipine, nitrendiplne, nicardipine, and so on). The chemical structures of these molecules reflect the considerable diversity in calcium channel inhibitors, and yet other types of calcium channel inhibitors will certainly be developed in the future. The receptors of all of the calcium channel in-
Volume 84 (suppl 1B)
SYMPOSIUM ON INDAPAMIDE--LAZDUNSKI
Ca 2÷ 0 0 000
B adrenergic agoni~st
~(~k
/ -- ----
~receptor ~ y l a t e c y c l a s e
~
Figure 3. Calcium channel modulation by beta agonists.
hibitors have now been identified [9]. The protein receptors of a given inhibitor are similar but not identical in the various cell types [6]. The diagram at the bottom of Figure 2 illustrates the very interesting result recently obtained in certain laboratories, including our own. It demonstrates that the efficacy of the dihydropyridines depends on the polarization of the smooth, cardiac, and skeletal muscles [10-12]. The doseresponse curve for inhibition of the calcium channel by molecules of the nifedipine/nitrendipine type is shifted towards the right when the cell is hyperpolarized and towards the left when the cell is depolarized. The more intensely the cell is depolarized, the more sensitive it is to inhibition by the dihydropyridines. This result is very important in the therapeutic context: because "diseased" cells are frequently more depolarized than healthy cells, this type of molecule would act preferentially on the depolarized diseased cells. In a healthy organism, the dihydropyridines tend to be more active in the least polarized cardiac cells or smooth muscle cells. We know, particularly on the basis of data obtained in cardiac cells, that the functioning of the calcium channel can be modulated. The efficacy of the calcium channel is modulated by beta-adrenergic agonists. The sequence of events leading to the modulation of the calcium channel in cardiac cells is represented in Figure 3. The beta-adrenergic agonist binds to its receptor. This receptor activates the adenylate cyclase system, which induces an increase in cyclic AMP inside the cell. Cyclic AMP activates a protein kinase that phosphorylates the calcium channel. As a result, the phosphorylated calcium channel becomes more active (with an increased probability of opening) and more calcium will enter the cell [3], inducing more contraction. This schematic representation explains the' action of beta-type antagonists. If the agonists are prevented from acting, the positive modulation of the activity of the calcium channel is also prevented, and cardiac contraction is decreased. January 29, 1988
/
(~(Z~ E
Ca 2* channel
~~)//~./~
ein kinase
This principle is also true for secretory cells, in which excitation-secretion coupling is due to the functioning of a calcium channel that can be modulated. In a secretory cell, whether it secretes neurotransmitters or hormones, there is also stimulation of the system (beta agonist leads to increased cyclic AMP, resulting in activation of the calcium channel). A beta antagonist decreases the efficiency of the calcium channel and, in principle, decreases secretion. This opens a considerable area for research into new antihypertensive agents. We have seen that it is possible to inhibit the calcium channel or to modulate the calcium channel by means of beta antagonists. It is also possible to activate one or several families of cellular potassium channels (Figure 4). Activation of the potassium channel of smooth muscle cells leads to the efflux of potassium and, consequently, to a state of hyperpolarization. This hyperpolarization widens the gap between the membrane potential and the threshold potential for opening of the calcium channel. The greater the difference between the resting membrane potential and the threshold potential for opening of the calcium channel, the lower is the probability of opening of the calcium channel-that is, the relaxation effect will be greater. INTRACELLULAR MEMBRANE SYSTEMS
The intracellular systems that regulate the cytoplasmic concentration of calcium are not found exclusively in cardiac cells or smooth muscle cells but are present in all cell types (Figure 5). The system of calcium storage correspondsto the endoplasmic reticulum in the majority of cells or the sarcoplasmic reticulum in muscle cells. The sarcoplasmic reticulum stores calcium obtained from t h e cytoplasm. This occurs in the presence of a gradient and therefore requires hydrolysis of ATP. The calcium stored in the sarcoplasmic reticulum can be released during contraction by means of a recently identified mechanism. The messenger that induces calcium release during contraction is inositol triphosphate [13]. The American Journal of Medicine
Volume 84 (suppl 1B)
5
SYMPOSIUM ON INDAPAMIDE--LAZDUNSKI
ACTIVATOR
Ca=*
" lo
7
K•
K" CHANNELACTIVATION~
HYPERPOLARIZATION~
DECREASEDPROBABILITYOF Ca~*CHANNELOPENING
Figure 4. One strategy to decrease contraction involves hyperpolarization of the smooth muscle cells to increase the difference between the resting potential and the threshold potential for activation of the calcium channel as much as possible.
~xagonists
Ca="~'~ Bag°nists
p.~.~l
~
~'~
~
i
p
Kinase J
~
~'- J
\
Phospholipase C
Diacylglycer~ol Inositol tri-I~hosphate
Ca 2o
ATP Ca="stores -- ~
s ,t
~~Phospholipase C AnglotenslnII
Inositol Triphosphate.
Inositol triphosphate is an internal messenger present in all cells. It is formed from a particular class of membrane phospholipids called phosphatidyl inositol. The intracellular formation of inositol triphosphate is linked to the action of a certain number of extracellular effectors--effectors of the alpha agonist type or effectors of the angiotensin type, for example (Figure 5). When an alpha agonist stimulates a smooth muscle cell, it binds to its receptor. This receptor binds to an enzyme called phospholipase C, by a complex process that will not be described here. This enzyme breaks down a particular class of lipids in the membrane, resulting in the formation of diacylglycerol and inositol triphosphate [13]. Inositol triphosphate, in turn, binds to its own intracellular receptor, resulting in the opening of a calcium channel, the release of calcium from the storage system, and consequent con-
6
Figure 5. Description and pharmacologic aspects of systems of intracellular calcium mobilization.
traction. The calcium channel activated by inositol triphosphate is entirely distinct from the cell membrane calcium channel described earlier. The same process can be described for angiotensin. The receptor for angiotensin II is linked to a phospholipase C; when angiotensin binds to its receptor, it activates phospholipase C, leading to the formation of diacylglycerol and inositol triphosphate. Inositol triphosphate binds to its own intracellular receptor, releases the stored calcium, and induces contraction. Pharmacologic Aspects. In regard to the pharmacologic aspects of these processes, it is clear that if the action of alpha agonists is inhibited by alpha antagonists, the receptor will no longer be stimulated, phospholipase C will no ionger be activated, inosito] triphosphate will no longer be produced, and calcium stored in the reticulum will not be released. This will result in reduced contraction.
January29, 1988 The AmericanJournalof Medicine Volume84 (suppl1B)
SYMPOSIUM ON INDAPAMIDE--LAZDUNSKI
Similarly, if the level of angiotensin II is decreased, less inositol triphosphate will be formed, less calcium will be released from the storage system, and contraction will be reduced. Consequently, any intervention that decreases the angiotensin concentration--whether in the form of drugs that inhibit the converting enzyme or another intermediate step in the production of this peptide, or systems that inhibit renin secretion--will therefore prevent the release of calcium from the storage system to the cytoplasm. In summary, to reduce hypertension, i.e., reduce excessive contraction, we can act on the plasma membrane calcium channel with calcium antagonists, we can modulate the cardiac calcium channel mediated by cyclic AMP with beta antagonists, or we can act on the inositol triphosphate system by using alpha-adrenergic antagonists, angiotensin antagonists, or any other system that prevents the formation of angiotensin. In the future, new molecules can be conceived to act on the intracellular metabolism of inositol triphosphate and/or on inositol triphosphate receptor recognition.
NH CO~ NH~C '#~ NH~ HN~I~N
NH7 Amiloride
Blocked
Na* N
by
~
Na*
Na*
5-10 mM
amiloride
/
/ Ca2. ~ 1 = ,
Na ÷
SODIUM TRANSPORT
140 mM
This discussion of sodium transport systems will lead us to another extremely important class of molecules used in the treatment of hypertension, the diuretics. In particular, we shall present those drugs for which the mechanism of action has been defined and which act on sodium transport mechanisms. The mechanisms of sodium transport presented in Figure 6 are those involved in cardiac cells and, with a few minor differences, in vascular cells. Efflux System. In every cell, the internal sodium concentrations are much lower than the external concentrations: about 140 mM in the extracellular environment and about 5 to 10 mM in the intracellular environment. As is the case with calcium, the maintenance of a low intracellular sodium concentration depends on the intervention of a system able to pump sodium from the interior of the cell to the exterior. This pump is an enzyme--sodium/potassium ATPase--which is the site of action of an important class of drugs, the digitalis glycosides [14,15]. Influx Systems. There are two major sodium influx systems in the cardiac cell and the vascular cell--the sodium/proton exchange system and sodium/potassium/ chloride co-transport system. The function of the sodium/ proton (sodium/hydrogen) exchange system is to take sodium into the cell in exchange for the expulsion of a proton. This is an important system in the regulation of the intracellular pH and it is present in all cells [14,15]. The sodium/proton exchange system can be analyzed relatively easily either by measuring the influx of sodium into the cell with radioactive sodium or by using flu6rometric measurement of variations in intracellular pH to determine the efflux of protons. The sodium/potassium/chloride cotransport system transports one sodium ion for one potas-
Blocked by furosemide, bumetanide. etc. H
I
CH,- ~
H Noi oo
Furosemide
Figure 6. Sodium transport systems.
slum ion and two chloride ions. This co-transport system plays an important role in the influx of sodium and potassium into the cell. In cardiac cells, for example, 50 percent of the potassium entering during steady-state conditions enters as a result of sodium/potassium/chloride co-transport (almost all of the rest enters via sodium/potassium ATPase) [16]. There is also another system allowing influx of sodium into the cell--the sodium/calcium exchange system described earlier. Pharmacologic Aspects. Studies conducted over recent years have demonstrated that the sodium/proton exchange system and the sodium/potassium/chloride cotransport system are the targets for two extremely important classes of diuretics. The sodium/proton exchange system is blocked by the amiloride class of diuretics [17]. The sodium/potassium/chloride co-transport system is blocked by a class of molecules belonging to the structural family of furosemide (bumetamide, benzetamide, and so on) [16]. Figure 7 schematically represents the action of
January 29, 1988 The American Journal of Medicine Volume 84 (suppl 1B)
7
SYMPOSIUM ON INDAPAMIDE~LAZDUNSKI
LUMEN
BLOOD
AMILORIDE K+ a÷
- Na÷
HCO; + H+ H+
1
#
CI-
C02
' CO2 /
J "L"',,~ Na+ / ,,I"
AMILORIDE
HCO;
- ATP
,_ Na+ K*
K+
Figure 7. level
Mechanism of action of amiloride at the renal
amiloride in the proximal and distal convoluted tubules of the kidneys. In the proximal convoluted tubules, sodium is resorbed as follows: sodium passes from the lumen towards the interior of the cell due to the action of the sodium/proton exchange system (the permeability of sodium is therefore related to regulation of intracellular pH and the extrusion of acid); after penetrating into the interior of the cell, sodium then enters the blood compartment due to the action of the sodium/potassium ATPase pump. In the distal convoluted tubules, the major sodium influx
pathway is the apical sodium channel in which sodium transport does not require exchange with a proton. Sodium enters the cell via the sodium channel and leaves the cell to enter the blood compartment via the sodium/ potassium ATPase pump, as in the proximal tubules. Amiloride blocks both the sodium/proton exchange system [17] and the apical sodium channel [18-20]. Amiloride therefore has two types of molecular targets, both of which transport sodium, but with different structures. Amiloride blocks the sodium/proton exchange system at concentrations measured in micromoles but acts on the apical sodium channel at concentrations about 10 to 100 times lower. These structures are completely different, as derivatives of amiloride that selectively act on the sodium/ proton exchange system without affecting the apical sodium channel and derivatives of amiloride that selectively act on the apical sodium channel without affecting the sodium/proton exchange system have been synthesized. When amiloride inhibits the influx of sodium, it also inhibits the efflux of potassium. The mechanism linking the system of influx of sodium via the sodium channel and the system of efflux of potassium is unknown, and the identity of this potassium channel and its corresponding specific pharmacologic features have yet to de determined. The family of molecules of the furosemide type acts on the sodium/potassium/chloride co-transport system, which is essentially located in the intermediate part of the nephron, the loop of Henle. This sodium/potassium/chloride system participates in sodium resorption but at a different site from the sodium/proton and sodium channel systems. In summary, we know only two major types of mechanisms of action for diuretics: those that correspond to the mechanism of action of amiloride and those that correspond to the mechanism of action of molecules of the bumetamide and furosemide type. The targets of these diuretics are present not only in the renal cells just described, but also in all cell types, including vascular smooth muscle cells. The molecular mechanisms of the thiazide diuretics or of the triamterene-type agents are unknown. In the case of indapamide, a non-thiazide diuretic with demonstrated vascular properties, the study of these molecular mechanisms warrants particular attention.
REFERENCES
1. Carafoli E, Caroni P: The calcium pumping ATPase of heart plasma membrane. In: BrautbarN, ed. Myocardialand skeletal muscle bioenergetics.New York:Plenum,1986; 563-572. 2. TsienRW: Calciumchannelsin excitablecell membranes. Annu Rev Physiol 1983; 45: 341-358. 3. Reuter H: Calcium channel modulation by neurotransmitters, enzymes and drugs. Nature 1983; 301: 569-574. 4. SperelakisN: Hormonal and neurotransmittersregulation of
8
January 29, 1988
The American Journal of Medicine
Ca' ' influxthrough voltage-dependentslow channels in cardiac muscle membrane. Membr Biochem 1984; 5: 131-166. Reuter H, Seitz N: The dependenceof calciumeffluxfrom cardiac muscle on temperature and externalion composition. J Physiol (Lond) 1968; 195: 451-470. 6. Schmid A, Barhanin J, Coppola T, Borsotto M, Lazdunski M: Immunochemical analysisof sub-unit structures of 1,4-dihydropyridine receptors associated with voltage dependent
Volume 84 (suppl 1B)
SYMPOSIUM ON INDAPAMIDE--LAZDUNSKI
7.
8.
9.
10.
11.
12.
Ca' ' channels in skeletal, cardiac and smooth muscles. Biochemistry 1986; 25: 3492-3495. Cooper CL, Vandaele S, Barhanin J, Fosset M, Lazdunski M, Hosey MM: Puritication and characterization of the dihydropyridine sensitive voltage-dependent calcium channel from cardiac tissue. J Biol Chem 1987; 262: 509-512. Vandaele S, Fosset M, Galizzi JP, Lazdunski M: Monoclonal antibodies that co-immuno precipitate the 1,4-dihydropyridine and phenylalkylamine binding components associated with the voltage-dependent Ca" ~ channels from skeletal muscle. Biochemistry 1987; 26: 5-9. Galizzi JP, Borsotto M, Barhanin J, Fosset M, Lazdunski M: Characterization and photoaffinity labelling of receptor sites for the Ca t* channel inhibitors d-cis-diltiazem, (+ - ) bepridil, ( - ) desmethoxyverapamil and (+) PN 200-110 in skeletal muscle transverse tubule membranes. J Biol Chem 1986; 261 : 1393-1397. Sanguinetti MC, Kass RS: Voltage-dependent block of calcium channel current in the calf cardiac Purkinje fiber by dihydropyridine calcium channel antagonists. Circ Res 1984; 55: 336-348. Bean BP: Nitrendipine block of cardiac calcium channels: highaffinity binding in the inactivated state. Proc Natl Acad Sci USA 1984; 81: 6388-6392. Cognard C, Romey G, Galizzi JP, Fosset M, Lazdunski M: Dihydropyridine sensitive Ca' ~ channels in mammalian skeletal muscle cells in culture: electrophysiol6gical properties and interactions with Ca' ~ channel activator (BK 8644) and inhibitor (PN 200-100). Proc Natl Acad Sci USA 1986; 83: 1518-
January 29, 1988
1522. Berridge MJ, Irvine RF: Inositol triphosphate, a novel second messenger in cellular signal transduction. Nature 1984; 312: 315-321. 14. Lazdunski M, Frelin C, Vigne P: The sodium/hydrogen exchange system in cardiac cells: its biochemical and pharmacological properties and its role in regulating internal concentrations of sodium and internal pH. J Mol Cell Cardiol 1985; 17: 1029-1042. 15. Frelin C, Vigne P, Lazdunski M: The cardiac Na'/H" exchange system. Its role in inotropy. In: Erdman E, Greff K, Skou JC, eds. Cardiac glycosides, 1785-1985. Darmstadt: Steinkopft Verlag, 1986; 207-213. 16. Frelin C, Chassande O, Lazdunski M: Biochemical characterization of Na'/K+/CI co-transport in chick cardiac cells. Biochem Biophys Res Commun 1986; 134: 326-331. 17. Vigne P, Frelin C, Cragoe EJ, Lazdunski M: Structure-activity relationships of amiloride and certain of its analogues in relation to the blockade of the Na'/H ~ exchange system. Mol Pharmacol 1984; 25: 131-136. 18. Lindermann B: Fluctuation analysis of sodium channels in epithelia. Annu Rev Physiol 1984; 46: 497-515. 19. Barbry P, Frelin C, Vigne P, Cragoe EJ, Lazdunski M: [3H] phenamil, a radiolabelled diuretic for the analysis of the amiloride-sensitive Na" channels in kidney membranes. Biochem Biophys Res Commun 1986; 135: 25-32. 20. Freiin C, Vigne P, Barbry P, Lazdunski M: Molecular properties of amiloride action and of its Na" transporting targets. Kidney Int 1986; 32: 785-793. 13.
The American Journal of Medicine
Volume 84 (suppl 1B)
9
MICHEL LAZDUNSKI, D.Sc. Nice, France
Various systems contribute to the regulation of intracellular calcium. The most important are: (1) the voltage-dependent calcium channel; (2) sodium/calcium exchange, resulting in a correlation between intracellular sodium and calcium; (3) plasma membrane calcium ATPase; and (4) the inositol triphosphate-regulated channel, releasing calcium from the endoplasmic and sarcoplasmic reticulum stores. The essential properties of these different systems, their pharmacologic features, and their regulation by various agonists (beta- and alpha-adrenergic angiotensin-type peptides, etc.) must be considered both for an understanding of the pathophysiology of hypertension and for its treatment. The aim of this study was to examine the various ionic transport systems --essentially those involved in sodium and calcium transport--that are the basis for smooth muscle and cardiac muscle contraction, and to study the molecular pharmacologic aspects of these transport systems.
CALCIUM TRANSPORT An absolutely fundamental point is that in every cell--not just cardiac or vascular cells--the external calcium concentration is much greater than the internal calcium concentration (Figure 1). The external concentration of ionized calcium is approximately 1.8 mM, whereas the cytoplasmic concentration, during the period of relaxation of the muscle cells, is about 0.1 /~M. A number of calcium transport systems are necessary to maintain this calcium gradient between the exterior and the interior of the cell.
EXTERNAL MEMBRANE SYSTEMS
From the Centre de Biochimiedu Centre National de la Recherche Scientifique (CNRS), Nice, France. Requestsfor reprintsshould be addressed to Professor Michel Lazdunski, Centre de Biochimie du CNRS, Parc Valrose, 06034 Nice Cedex, France.
The systems represented in Figure 1 are calcium transport systems located in the plasma membrane of cardiac and smooth muscle cells. Efflux System. The major system of efflux of calcium from the interior of the cell towards the exterior is not specific to smooth muscle cells or to cardiac cells, but is present in all cells. Calcium ions are taken from the interior of the cell and are expelled towards the exterior. As this operation occurs in the presence of an ionic gradient, it requires the hydrolysis of ATP, which explains the necessary intervention of an enzyme known as calcium ATPase [1]. Influx Systems. There are two systems of calcium influx in the plasma membrane. The first is the calcium channel, which represents the major system of calcium influx in smooth muscle and cardiac muscle plasma membranes [2-4]. This calcium channel will be discussed in more detail in relation to calcium channel inhibitors. The other calcium influx system in the plasma membrane, in reality, can be used either as an influx sys-
January 29, 1988 The American Journal of Medicine Volume84 (suppl 1B)
3
SYMPOSIUMON INDAPAMIDE--LAZDUNSKI
Blockedby Ca2" channel blockers
C a ;t+
/ °-°;°
--,~
Ca 2"
÷ + + ++ - - ~ - __~ __~ _ _ ~ " - ) ~ _ _ ~ l ~
+ -
Oiiw eleCgrr°~l . cihaeenr~ lj'
1.8 mM
polarizedmembrane (normalstate} "
/
[Ca2"1,=0.1 pM
• depolarized membrane
\
, \oo .a@
CH1~cHICH~
CH3
,
1
H~CO---I~'I~---"l m C - - (CHT)~ - N -
II
H3C0R'~ ~
J
Nokoown
pharmacology
I
Ca
(CH;,)~.---l;~l~
OCH~
~
OCH~
I
C-- N
Verapamll .
T~7 •
(R--H); D600 : (R--0CH3)
2.
0Me
I I I
Weakly blocked by amiloridederivatives Figure 1. Systems regulating calcium transport in the plasma membrane of cardiac and vascular cells.
I
o
CH~-CHe-NMe e Diltlazem
tern or as an efflux system. This system of reversible ex, change, which links the efflux of sodium with the influx of calcium or vice versa, is called the sodium/calcium exchange system [5]. Pharmacologic Aspects. On the basis of this schematic representation, it is possible to imagine a variety of molecules that coutd be active in hypertension. A molecute could stimulate calcium ATPase, i.e., expel calcium from the cell in order to induce less contraction; however, at the present time, the pharmacology of calcium ATPase is unknown. A molecule could be an effector of the sodium/ calcium exchange system, but the pharmacology of this exchange has not been well defined (although it can be blocked by substitution of sodium with lithium and, to a lesser degree, by certain derivatives of amiloride). In contrast, the pharmacology of the calcium channels has been perfectly defined. Figure 2 summarizes a number of data concerning calcium channel inhibitors. At the top of Figure 2, a diagram explains the functioning of the plasma membrane calcium channel. This calcium channel is a protein that has now been purified and its structure clearly identified [6-8]. This protein pore can be either closed or open and therefore permeable to calcium, depending on the membrane potential. It opens during the action potential and allows influx of calcium, which subsequently induces contraction. The calcium channel is blocked by a whole series of inhibitors, the principal examples of which are presented in the center of Figure 2. These are molecules of the verapamil type, the diltiazem type, and the dihydropyridine type (ni-
4
January 29, 1988
The American Journal of Medicine
~N07
[ ~
1
NO~
I
H
H
Nlfedlplne
Nitrendiplne
DEPOLARIZED 1
Hy[~r~ pERpo E.=~O~=. LARIZ ..~=D Ko~= 13nM
E .5 Z Kos=0.15nM • 0 L . . , -12 -11 -10 -9 -8 -7 -6 -5 Log [ (+) PN200-- 110] Figure 2. Pharmacologic aspects ofthe calcium channels (reproduced from [12]).
fedipine, nitrendiplne, nicardipine, and so on). The chemical structures of these molecules reflect the considerable diversity in calcium channel inhibitors, and yet other types of calcium channel inhibitors will certainly be developed in the future. The receptors of all of the calcium channel in-
Volume 84 (suppl 1B)
SYMPOSIUM ON INDAPAMIDE--LAZDUNSKI
Ca 2÷ 0 0 000
B adrenergic agoni~st
~(~k
/ -- ----
~receptor ~ y l a t e c y c l a s e
~
Figure 3. Calcium channel modulation by beta agonists.
hibitors have now been identified [9]. The protein receptors of a given inhibitor are similar but not identical in the various cell types [6]. The diagram at the bottom of Figure 2 illustrates the very interesting result recently obtained in certain laboratories, including our own. It demonstrates that the efficacy of the dihydropyridines depends on the polarization of the smooth, cardiac, and skeletal muscles [10-12]. The doseresponse curve for inhibition of the calcium channel by molecules of the nifedipine/nitrendipine type is shifted towards the right when the cell is hyperpolarized and towards the left when the cell is depolarized. The more intensely the cell is depolarized, the more sensitive it is to inhibition by the dihydropyridines. This result is very important in the therapeutic context: because "diseased" cells are frequently more depolarized than healthy cells, this type of molecule would act preferentially on the depolarized diseased cells. In a healthy organism, the dihydropyridines tend to be more active in the least polarized cardiac cells or smooth muscle cells. We know, particularly on the basis of data obtained in cardiac cells, that the functioning of the calcium channel can be modulated. The efficacy of the calcium channel is modulated by beta-adrenergic agonists. The sequence of events leading to the modulation of the calcium channel in cardiac cells is represented in Figure 3. The beta-adrenergic agonist binds to its receptor. This receptor activates the adenylate cyclase system, which induces an increase in cyclic AMP inside the cell. Cyclic AMP activates a protein kinase that phosphorylates the calcium channel. As a result, the phosphorylated calcium channel becomes more active (with an increased probability of opening) and more calcium will enter the cell [3], inducing more contraction. This schematic representation explains the' action of beta-type antagonists. If the agonists are prevented from acting, the positive modulation of the activity of the calcium channel is also prevented, and cardiac contraction is decreased. January 29, 1988
/
(~(Z~ E
Ca 2* channel
~~)//~./~
ein kinase
This principle is also true for secretory cells, in which excitation-secretion coupling is due to the functioning of a calcium channel that can be modulated. In a secretory cell, whether it secretes neurotransmitters or hormones, there is also stimulation of the system (beta agonist leads to increased cyclic AMP, resulting in activation of the calcium channel). A beta antagonist decreases the efficiency of the calcium channel and, in principle, decreases secretion. This opens a considerable area for research into new antihypertensive agents. We have seen that it is possible to inhibit the calcium channel or to modulate the calcium channel by means of beta antagonists. It is also possible to activate one or several families of cellular potassium channels (Figure 4). Activation of the potassium channel of smooth muscle cells leads to the efflux of potassium and, consequently, to a state of hyperpolarization. This hyperpolarization widens the gap between the membrane potential and the threshold potential for opening of the calcium channel. The greater the difference between the resting membrane potential and the threshold potential for opening of the calcium channel, the lower is the probability of opening of the calcium channel-that is, the relaxation effect will be greater. INTRACELLULAR MEMBRANE SYSTEMS
The intracellular systems that regulate the cytoplasmic concentration of calcium are not found exclusively in cardiac cells or smooth muscle cells but are present in all cell types (Figure 5). The system of calcium storage correspondsto the endoplasmic reticulum in the majority of cells or the sarcoplasmic reticulum in muscle cells. The sarcoplasmic reticulum stores calcium obtained from t h e cytoplasm. This occurs in the presence of a gradient and therefore requires hydrolysis of ATP. The calcium stored in the sarcoplasmic reticulum can be released during contraction by means of a recently identified mechanism. The messenger that induces calcium release during contraction is inositol triphosphate [13]. The American Journal of Medicine
Volume 84 (suppl 1B)
5
SYMPOSIUM ON INDAPAMIDE--LAZDUNSKI
ACTIVATOR
Ca=*
" lo
7
K•
K" CHANNELACTIVATION~
HYPERPOLARIZATION~
DECREASEDPROBABILITYOF Ca~*CHANNELOPENING
Figure 4. One strategy to decrease contraction involves hyperpolarization of the smooth muscle cells to increase the difference between the resting potential and the threshold potential for activation of the calcium channel as much as possible.
~xagonists
Ca="~'~ Bag°nists
p.~.~l
~
~'~
~
i
p
Kinase J
~
~'- J
\
Phospholipase C
Diacylglycer~ol Inositol tri-I~hosphate
Ca 2o
ATP Ca="stores -- ~
s ,t
~~Phospholipase C AnglotenslnII
Inositol Triphosphate.
Inositol triphosphate is an internal messenger present in all cells. It is formed from a particular class of membrane phospholipids called phosphatidyl inositol. The intracellular formation of inositol triphosphate is linked to the action of a certain number of extracellular effectors--effectors of the alpha agonist type or effectors of the angiotensin type, for example (Figure 5). When an alpha agonist stimulates a smooth muscle cell, it binds to its receptor. This receptor binds to an enzyme called phospholipase C, by a complex process that will not be described here. This enzyme breaks down a particular class of lipids in the membrane, resulting in the formation of diacylglycerol and inositol triphosphate [13]. Inositol triphosphate, in turn, binds to its own intracellular receptor, resulting in the opening of a calcium channel, the release of calcium from the storage system, and consequent con-
6
Figure 5. Description and pharmacologic aspects of systems of intracellular calcium mobilization.
traction. The calcium channel activated by inositol triphosphate is entirely distinct from the cell membrane calcium channel described earlier. The same process can be described for angiotensin. The receptor for angiotensin II is linked to a phospholipase C; when angiotensin binds to its receptor, it activates phospholipase C, leading to the formation of diacylglycerol and inositol triphosphate. Inositol triphosphate binds to its own intracellular receptor, releases the stored calcium, and induces contraction. Pharmacologic Aspects. In regard to the pharmacologic aspects of these processes, it is clear that if the action of alpha agonists is inhibited by alpha antagonists, the receptor will no longer be stimulated, phospholipase C will no ionger be activated, inosito] triphosphate will no longer be produced, and calcium stored in the reticulum will not be released. This will result in reduced contraction.
January29, 1988 The AmericanJournalof Medicine Volume84 (suppl1B)
SYMPOSIUM ON INDAPAMIDE--LAZDUNSKI
Similarly, if the level of angiotensin II is decreased, less inositol triphosphate will be formed, less calcium will be released from the storage system, and contraction will be reduced. Consequently, any intervention that decreases the angiotensin concentration--whether in the form of drugs that inhibit the converting enzyme or another intermediate step in the production of this peptide, or systems that inhibit renin secretion--will therefore prevent the release of calcium from the storage system to the cytoplasm. In summary, to reduce hypertension, i.e., reduce excessive contraction, we can act on the plasma membrane calcium channel with calcium antagonists, we can modulate the cardiac calcium channel mediated by cyclic AMP with beta antagonists, or we can act on the inositol triphosphate system by using alpha-adrenergic antagonists, angiotensin antagonists, or any other system that prevents the formation of angiotensin. In the future, new molecules can be conceived to act on the intracellular metabolism of inositol triphosphate and/or on inositol triphosphate receptor recognition.
NH CO~ NH~C '#~ NH~ HN~I~N
NH7 Amiloride
Blocked
Na* N
by
~
Na*
Na*
5-10 mM
amiloride
/
/ Ca2. ~ 1 = ,
Na ÷
SODIUM TRANSPORT
140 mM
This discussion of sodium transport systems will lead us to another extremely important class of molecules used in the treatment of hypertension, the diuretics. In particular, we shall present those drugs for which the mechanism of action has been defined and which act on sodium transport mechanisms. The mechanisms of sodium transport presented in Figure 6 are those involved in cardiac cells and, with a few minor differences, in vascular cells. Efflux System. In every cell, the internal sodium concentrations are much lower than the external concentrations: about 140 mM in the extracellular environment and about 5 to 10 mM in the intracellular environment. As is the case with calcium, the maintenance of a low intracellular sodium concentration depends on the intervention of a system able to pump sodium from the interior of the cell to the exterior. This pump is an enzyme--sodium/potassium ATPase--which is the site of action of an important class of drugs, the digitalis glycosides [14,15]. Influx Systems. There are two major sodium influx systems in the cardiac cell and the vascular cell--the sodium/proton exchange system and sodium/potassium/ chloride co-transport system. The function of the sodium/ proton (sodium/hydrogen) exchange system is to take sodium into the cell in exchange for the expulsion of a proton. This is an important system in the regulation of the intracellular pH and it is present in all cells [14,15]. The sodium/proton exchange system can be analyzed relatively easily either by measuring the influx of sodium into the cell with radioactive sodium or by using flu6rometric measurement of variations in intracellular pH to determine the efflux of protons. The sodium/potassium/chloride cotransport system transports one sodium ion for one potas-
Blocked by furosemide, bumetanide. etc. H
I
CH,- ~
H Noi oo
Furosemide
Figure 6. Sodium transport systems.
slum ion and two chloride ions. This co-transport system plays an important role in the influx of sodium and potassium into the cell. In cardiac cells, for example, 50 percent of the potassium entering during steady-state conditions enters as a result of sodium/potassium/chloride co-transport (almost all of the rest enters via sodium/potassium ATPase) [16]. There is also another system allowing influx of sodium into the cell--the sodium/calcium exchange system described earlier. Pharmacologic Aspects. Studies conducted over recent years have demonstrated that the sodium/proton exchange system and the sodium/potassium/chloride cotransport system are the targets for two extremely important classes of diuretics. The sodium/proton exchange system is blocked by the amiloride class of diuretics [17]. The sodium/potassium/chloride co-transport system is blocked by a class of molecules belonging to the structural family of furosemide (bumetamide, benzetamide, and so on) [16]. Figure 7 schematically represents the action of
January 29, 1988 The American Journal of Medicine Volume 84 (suppl 1B)
7
SYMPOSIUM ON INDAPAMIDE~LAZDUNSKI
LUMEN
BLOOD
AMILORIDE K+ a÷
- Na÷
HCO; + H+ H+
1
#
CI-
C02
' CO2 /
J "L"',,~ Na+ / ,,I"
AMILORIDE
HCO;
- ATP
,_ Na+ K*
K+
Figure 7. level
Mechanism of action of amiloride at the renal
amiloride in the proximal and distal convoluted tubules of the kidneys. In the proximal convoluted tubules, sodium is resorbed as follows: sodium passes from the lumen towards the interior of the cell due to the action of the sodium/proton exchange system (the permeability of sodium is therefore related to regulation of intracellular pH and the extrusion of acid); after penetrating into the interior of the cell, sodium then enters the blood compartment due to the action of the sodium/potassium ATPase pump. In the distal convoluted tubules, the major sodium influx
pathway is the apical sodium channel in which sodium transport does not require exchange with a proton. Sodium enters the cell via the sodium channel and leaves the cell to enter the blood compartment via the sodium/ potassium ATPase pump, as in the proximal tubules. Amiloride blocks both the sodium/proton exchange system [17] and the apical sodium channel [18-20]. Amiloride therefore has two types of molecular targets, both of which transport sodium, but with different structures. Amiloride blocks the sodium/proton exchange system at concentrations measured in micromoles but acts on the apical sodium channel at concentrations about 10 to 100 times lower. These structures are completely different, as derivatives of amiloride that selectively act on the sodium/ proton exchange system without affecting the apical sodium channel and derivatives of amiloride that selectively act on the apical sodium channel without affecting the sodium/proton exchange system have been synthesized. When amiloride inhibits the influx of sodium, it also inhibits the efflux of potassium. The mechanism linking the system of influx of sodium via the sodium channel and the system of efflux of potassium is unknown, and the identity of this potassium channel and its corresponding specific pharmacologic features have yet to de determined. The family of molecules of the furosemide type acts on the sodium/potassium/chloride co-transport system, which is essentially located in the intermediate part of the nephron, the loop of Henle. This sodium/potassium/chloride system participates in sodium resorption but at a different site from the sodium/proton and sodium channel systems. In summary, we know only two major types of mechanisms of action for diuretics: those that correspond to the mechanism of action of amiloride and those that correspond to the mechanism of action of molecules of the bumetamide and furosemide type. The targets of these diuretics are present not only in the renal cells just described, but also in all cell types, including vascular smooth muscle cells. The molecular mechanisms of the thiazide diuretics or of the triamterene-type agents are unknown. In the case of indapamide, a non-thiazide diuretic with demonstrated vascular properties, the study of these molecular mechanisms warrants particular attention.
REFERENCES
1. Carafoli E, Caroni P: The calcium pumping ATPase of heart plasma membrane. In: BrautbarN, ed. Myocardialand skeletal muscle bioenergetics.New York:Plenum,1986; 563-572. 2. TsienRW: Calciumchannelsin excitablecell membranes. Annu Rev Physiol 1983; 45: 341-358. 3. Reuter H: Calcium channel modulation by neurotransmitters, enzymes and drugs. Nature 1983; 301: 569-574. 4. SperelakisN: Hormonal and neurotransmittersregulation of
8
January 29, 1988
The American Journal of Medicine
Ca' ' influxthrough voltage-dependentslow channels in cardiac muscle membrane. Membr Biochem 1984; 5: 131-166. Reuter H, Seitz N: The dependenceof calciumeffluxfrom cardiac muscle on temperature and externalion composition. J Physiol (Lond) 1968; 195: 451-470. 6. Schmid A, Barhanin J, Coppola T, Borsotto M, Lazdunski M: Immunochemical analysisof sub-unit structures of 1,4-dihydropyridine receptors associated with voltage dependent
Volume 84 (suppl 1B)
SYMPOSIUM ON INDAPAMIDE--LAZDUNSKI
7.
8.
9.
10.
11.
12.
Ca' ' channels in skeletal, cardiac and smooth muscles. Biochemistry 1986; 25: 3492-3495. Cooper CL, Vandaele S, Barhanin J, Fosset M, Lazdunski M, Hosey MM: Puritication and characterization of the dihydropyridine sensitive voltage-dependent calcium channel from cardiac tissue. J Biol Chem 1987; 262: 509-512. Vandaele S, Fosset M, Galizzi JP, Lazdunski M: Monoclonal antibodies that co-immuno precipitate the 1,4-dihydropyridine and phenylalkylamine binding components associated with the voltage-dependent Ca" ~ channels from skeletal muscle. Biochemistry 1987; 26: 5-9. Galizzi JP, Borsotto M, Barhanin J, Fosset M, Lazdunski M: Characterization and photoaffinity labelling of receptor sites for the Ca t* channel inhibitors d-cis-diltiazem, (+ - ) bepridil, ( - ) desmethoxyverapamil and (+) PN 200-110 in skeletal muscle transverse tubule membranes. J Biol Chem 1986; 261 : 1393-1397. Sanguinetti MC, Kass RS: Voltage-dependent block of calcium channel current in the calf cardiac Purkinje fiber by dihydropyridine calcium channel antagonists. Circ Res 1984; 55: 336-348. Bean BP: Nitrendipine block of cardiac calcium channels: highaffinity binding in the inactivated state. Proc Natl Acad Sci USA 1984; 81: 6388-6392. Cognard C, Romey G, Galizzi JP, Fosset M, Lazdunski M: Dihydropyridine sensitive Ca' ~ channels in mammalian skeletal muscle cells in culture: electrophysiol6gical properties and interactions with Ca' ~ channel activator (BK 8644) and inhibitor (PN 200-100). Proc Natl Acad Sci USA 1986; 83: 1518-
January 29, 1988
1522. Berridge MJ, Irvine RF: Inositol triphosphate, a novel second messenger in cellular signal transduction. Nature 1984; 312: 315-321. 14. Lazdunski M, Frelin C, Vigne P: The sodium/hydrogen exchange system in cardiac cells: its biochemical and pharmacological properties and its role in regulating internal concentrations of sodium and internal pH. J Mol Cell Cardiol 1985; 17: 1029-1042. 15. Frelin C, Vigne P, Lazdunski M: The cardiac Na'/H" exchange system. Its role in inotropy. In: Erdman E, Greff K, Skou JC, eds. Cardiac glycosides, 1785-1985. Darmstadt: Steinkopft Verlag, 1986; 207-213. 16. Frelin C, Chassande O, Lazdunski M: Biochemical characterization of Na'/K+/CI co-transport in chick cardiac cells. Biochem Biophys Res Commun 1986; 134: 326-331. 17. Vigne P, Frelin C, Cragoe EJ, Lazdunski M: Structure-activity relationships of amiloride and certain of its analogues in relation to the blockade of the Na'/H ~ exchange system. Mol Pharmacol 1984; 25: 131-136. 18. Lindermann B: Fluctuation analysis of sodium channels in epithelia. Annu Rev Physiol 1984; 46: 497-515. 19. Barbry P, Frelin C, Vigne P, Cragoe EJ, Lazdunski M: [3H] phenamil, a radiolabelled diuretic for the analysis of the amiloride-sensitive Na" channels in kidney membranes. Biochem Biophys Res Commun 1986; 135: 25-32. 20. Freiin C, Vigne P, Barbry P, Lazdunski M: Molecular properties of amiloride action and of its Na" transporting targets. Kidney Int 1986; 32: 785-793. 13.
The American Journal of Medicine
Volume 84 (suppl 1B)
9