**2. Ca2+ channels: Physiological role and pharmacological modification**

Rise in intracellular Ca2+ triggers a variety of physiological processes, and there are many channels and pumps involved in controlling intracellular Ca2+ level. Among them, voltage-

Dual L/N-Type Ca2+ Channel Blocker: Cilnidipine as a New Type of Antihypertensive Drug 31

a) translocation of the synaptic vesicle filled with norepinephrine and epinephrine to the active zone; b) docking at the active zone and priming; c) fusion triggered by Ca2+ and exocytosis; d) endocytosis of empty synaptic vesicle to become coated vesicles; e) endosome fusion and budding to make the synaptic vesicle re-generation; and f) the neurotransmitters uptake to the regenerated synaptic vesicle. Entered Ca2+ through voltage-gated Ca2+ channels bind to synaplotagmin, changing the conformation of a large protein superfamily called as SNARE complexes (formed by 4 α-helices; synaptobrevin, synaptotagmin, syntaxin and SNAP 25), leading to fusion of vesicle membrane into plasma membrane. At these steps, the N-type Ca2+ channel plays an important role as a Ca2+ supplier. More than 60 Ca2+

In the sympathetic nervous systems, N-type Ca2+ channels are localized at the nerve endings. Using a patch clamp method, N-type Ca2+ channels are shown to contribute about 85% of all other types of Ca2+ channels in the sympathetic neuronal cells. N-type Ca2+ channels have been demonstrated to predominantly regulate norepinephrine release in the superior cervical ganglia neurons using a selective N-type Ca2+ channel blocker ω-conotoxin GVIA (Hirning et al., 1988). This finding was further supported by subsequent experiments using isolated rat arterial preparations. In a clinical study, systemic administration of ω-

Chromaffin cells in the medulla of the adrenal gland are innervated by the splanchnic nerve and secrete catecholamines into the blood stream. In anesthetized dogs, the splanchnic nerve stimulation increased the catecholamine secretion from adrenal gland, which was effectively inhibited by an N-type Ca2+ channel blocker ω-conotoxin GVIA but not by L-type Ca2+ channel blockers nifedipine or verapamil (Kimura et al., 1994). Many in vitro studies also support that N-type Ca2+ channels are localized at chromaffin cells to regulate the release of catecholamine. Recently, it is shown that N-type Ca2+ channels are also localized at the human adrenocortical cells, playing an important role in the secretion of adrenocortical hormones (Aritomi et al., 2011a). Furthermore, N-type Ca2+ channels may offer the different way of controlling corticosteroid production in adrenocortical cells than other types of

Cilnidipine is a unique dihydropyridine derivative L-type Ca2+ channel blocker with an inhibitory action on the sympathetic N-type Ca2+ channels. As shown in Fig. 1, Ca2+ channels are ordinarily activated by membrane depolarization in the vascular cells or sympathetic neurons, leading to vascular contraction or neurotransmitter releases. During antihypertensive therapies with pure L-type Ca2+ channel blockers like nifedipine, the sympathetic reflex is sometimes occurred due to hypotension, leading to activation of sympathetic N-type Ca2+ channels, which induces several cardiovascular responses including vascular contraction, tachycardia and renin secretion (Takahara 2009). Cilnidipine can directly inhibit the sympathetic neurotransmitter release by its N-type Ca2+ channelblocking property, which may reduce risk of cardiovascular diseases closely associated with sympathetic nerve activation. The wide variety of pharmacological actions of cilnidipine has

channels open for each vesicle for rapid release (Uneyama et al., 1999).

conotoxin MVIIA (SNX-111) has been shown to induce sympatholytic action.

**3. New generation Ca2+ channel blocker: Cilnidipine** 

been investigated, which is summarized in Table 1.

**2.4 Ca2+ channels in the adrenal gland** 

voltage-gated Ca2+ channels.

gated Ca2+ channels play a key role in this process, which is a pharmacological target molecule for so-called Ca2+ channel blockers. In excitatory cells such as smooth and cardiac muscle cells and neurons, high voltage-activated (HVA) Ca2+ channels are well known to regulate a variety of cellular events, which include muscle contraction, neuronal electrical activity, the release of neurotransmitters and hormones as well as gene expressions. On the other hand, low voltage-activated (LVA) Ca2+ channels are expressed throughout the body, including nervous tissue, heart, kidney, smooth muscle and many endocrine organs. The channels in the brain are considered to be involved in repetitive low threshold firing and nociception. In the heart, they are expressed in the sino-atrial node and are considered to participate in cardiac pacemaking (Tanaka & Shigenobu, 2005).

## **2.1 Classification of Ca2+ channels**

Ca2+ channels are classified into at least 6 subtypes; namely, L-, N-, P-, Q-, R-, and T-type, based on electrophysiological and pharmacological evidences (Varadi et al., 1995). The Ttype Ca2+ channels are known as low-voltage-activated Ca2+ channels that activate and deactivate slowly, but inactivate rapidly. The other five types of Ca2+ channels are all highvoltage-activated Ca2+ channels, which depolarize at approximately –40 mV. Molecular biological techniques have shown that Ca2+ channels are composed of α1, α2-δ, β, and γ subunits using L-type Ca2+ channels from skeletal muscles. In particular, the αl subunit forms the Ca2+ transmission pore, which fulfills the most important function. Furthermore, 10 α1 subunits have been cloned and classified into 3 subfamilies: Cav1.x; Cav2.x; and Cav3.x, based on their gene sequence similarity (Catterall et al., 2003). More importantly, the α1 subunit has a binding site for Ca2+ channel blockers.

#### **2.2 Ca2+ channels in the cardiovascular system**

In the cardiovascular system, L-type Ca2+ channels are predominantly expressed in the heart and vessels, which regulate cardiac contractility, sinus nodal function and vascular tone. βadrenergic stimulation enhances the force of cardiac contraction through activation of cAMPmediated activation of protein kinase A that in turn increases the L-type Ca2+ channel currents, causing a greater rate of release of Ca2+ from the sarcoplasmic reticulum. In the arterial vessels, receptor stimulation or membrane depolarization activates Ca2+ influx through Ca2+ channels and myosin light chain kinase, leading to smooth muscle contraction. Thus, L-type Ca2+ channels have been recognized as a pharmacological target for the treatment of cardiovascular disease. Most of Ca2+ channel blockers are well known to have selectivity for vascular tissues rather than cardiac function. On the other hand, verapamil, diltiazem and bepridil have been shown to possess less vascular selectivity, which are used for supraventricular and/or ventricular arrhythmias. The selectivity of Ca2+ channel blockers for cardiac and vascular actions may be associated with that the membrane potential in vascular smooth muscle cells is definitely less negative than the diastolic membrane potential of working cardiac muscle cells. In the vascular system, arterioles appear to be more sensitive to Ca2+ channel blockers than venules; orthostatic hypotension is not a common adverse effect.

#### **2.3 Ca2+ channels in the sympathetic nerve system**

The most thoroughly characterized role of Ca2+ in the nerve is the triggering of exocytosis. The synaptic vesicle cycles at nerve endings could be divided into the following processes: a) translocation of the synaptic vesicle filled with norepinephrine and epinephrine to the active zone; b) docking at the active zone and priming; c) fusion triggered by Ca2+ and exocytosis; d) endocytosis of empty synaptic vesicle to become coated vesicles; e) endosome fusion and budding to make the synaptic vesicle re-generation; and f) the neurotransmitters uptake to the regenerated synaptic vesicle. Entered Ca2+ through voltage-gated Ca2+ channels bind to synaplotagmin, changing the conformation of a large protein superfamily called as SNARE complexes (formed by 4 α-helices; synaptobrevin, synaptotagmin, syntaxin and SNAP 25), leading to fusion of vesicle membrane into plasma membrane. At these steps, the N-type Ca2+ channel plays an important role as a Ca2+ supplier. More than 60 Ca2+ channels open for each vesicle for rapid release (Uneyama et al., 1999).

In the sympathetic nervous systems, N-type Ca2+ channels are localized at the nerve endings. Using a patch clamp method, N-type Ca2+ channels are shown to contribute about 85% of all other types of Ca2+ channels in the sympathetic neuronal cells. N-type Ca2+ channels have been demonstrated to predominantly regulate norepinephrine release in the superior cervical ganglia neurons using a selective N-type Ca2+ channel blocker ω-conotoxin GVIA (Hirning et al., 1988). This finding was further supported by subsequent experiments using isolated rat arterial preparations. In a clinical study, systemic administration of ωconotoxin MVIIA (SNX-111) has been shown to induce sympatholytic action.
