**4. Discussion**

120 min after DD (**Table 4**). Decrease of epinephrine plasma concentration by verapamil

Verapamil *i.c.v.* premedication caused that increase of plasma norepinephrine concentration after 5 min DD, increased from average 1.29 ± 0.22 in control to 1.58 ± 0.84 nM L−1, during 120 min after DD, but not from 1.29 ± 0.23 in control to 2.50 ± 0.42 nM L−1 (+98.56%), during 120 min after DD. Decrease in norepinephrine concentration by verapamil premedication was

0.9% NaCl (100 μL) 0.98 ± 0.00 0.75 ± 0.00 0.82 ± 0.00 0.71 ± 0.00 0.85 ± 0.11 0.85 ± 0.00 0.99 ± 0.12 DD40 0.34 ± 0.12 2.42 ± 0.09 3.15 ± 0.68 3.45 ± 0.53 3.38 ± 1.82 2.58 ± 1.13 2.87 ± 0.65 Diltiazem + DD40 0.70 ± 0.12 0.98 ± 0.42 0.99 ± 0.25 0.88 ± 0.10 1.01 ± 0.20 0.88 ± 0.12 0.56 ± 0.23 Nifedipine + DD40 0.90 ± 0.12 0.97 ± 0.14 0.89 ± 0.25 1.31 ± 0.58 1.41 ± 0.38 0.99 ± 0.18 0.90 ± 0.41 Verapamil + DD40 1.19 ± 0.29 1.34 ± 0.31 1.30 ± 0.28 1.34 ± 0.26 1.24 ± 0.59 1.39 ± 0.21 1.58 ± 0.35

0.9% NaCl (100 μL) 1.30 ± 0.16 1.32 ± 0.15 1.37 ± 0.48 1.36 ± 0.19 1.33 ± 0.58 1.33 ± 0.44 1.28 ± 0.17 DD40 1.29 ± 0.23 2.85 ± 0.48 2.93 ± 0.51 2.94 ± 0.76 2.31 ± 0.30 2.11 ± 0.32 2.32 ± 0.24 Diltiazem + DD40 0.82 ± 0.20 0.61 ± 0.31 0.83 ± 0.13 0.83 ± 0.33 1.12 ± 0.33 0.89 ± 0.12 0.69 ± 0.04 Nifedipine + DD40 0.95 ± 0.06 1.01 ± 0.14 0.99 ± 0.28 1.12 ± 0.38 1.41 ± 1.22 1.12 ± 0.72 1.43 ± 1.23 Verapamil + DD40 1.29 ± 0.22 1.94 ± 0.66 1.52 ± 0.42 1.63 ± 1.12 1.68 ± 1.10 1.42 ± 0.48 1.32 ± 0.24

0.9% NaCl (100 μL) 1.23 ± 0.08 1.24 ± 0.06 1.26 ± 0.10 1.24 ± 0.09 1.25 ± 0.12 1.31 ± 0.01 1.15 ± 0.07 DD40 0.93 ± 0.02 1.26 ± 0.01 2.18 ± 0.30 2.51 ± 0.52 2.25 ± 0.52 2.10 ± 0.35 2.33 ± 1.16 Diltiazem + DD40 0.99 ± 0.09 0.75 ± 0.51 0.53 ± 0.18 1.05 ± 0.26 0.98 ± 0.25 0.86 ± 0.53 0.74 ± 0.19 Nifedipine + DD40 0.96 ± 0.02 1.29 ± 0.12 1.53 ± 0.42 1.62 ± 0.28 2.52 ± 1.41 1.32 ± 0.19 1.02 ± 0.32 Verapamil + DD40 0.92 ± 0.04 1.51 ± 0.12 1.63 ± 0.09 1.51 ± 0.08 1.50 ± 0.12 1.53 ± 0.13 1.48 ± 0.25

**Table 4.** Comparative analysis *i.c.v.* diltiazem, nifedipine, and verapamil (in the dose of 1.0 or 2.0 mg *in toto*)

in sheep in comparison to the control values (ẍ ± SEM; n = 6).

premedication influence and DD40 on concentration epinephrine, norepinephrine and dopamine plasma level changes

*I.C.V.* verapamil premedication caused that plasma dopamine concentration after 5 min DD increased from 0.92 ± 0.04 in control to 1.52 ± 0.13 nM L−1, during 120 min after DD (+65.04%),

**0 5 10 15 30 60 120**

premedication was 753.08%.

236 Pain Relief - From Analgesics to Alternative Therapies

**Catecholamine Time (in min)**

75.71%.

*Epinephrine*

*Norepinephrine*

*Dopamine*

The results of this experiment showed that 1 min diltiazem, nifedipine, and/or verapamil (VGCCIs) *i.c.v.* infusion in doses 0.25, 0.5, 1.0, or 2.0 mg *in toto*, given 10 min before DD, decreased the intensity of visceral nocifensive responses, such as behavioral changes, tachycardia, hyperventilation, reticulo-ruminal motility inhibition, and efficiently prevented the appearance of cortisol and catecholamine concentration in the blood plasma, after two higher doses. It was established that these results revealed that the development and persistence of acute duodenal pain depends on the activation of Ca2+ ion flux, leading to neurohormones release and modulation of membrane excitability. It seems that VGCCIs given *i.c.v.* 10 min prior to DD, which was evoked by the darting pain, blocked specific receptors α(1) subunits of voltage-gated calcium channels in effector tissues, attenuate depolarization of cellular membranes, and liberation of neurotransducers important for pain perception in small ruminants. The confirmed analgesic effect of L-type VGCCIs proposes that these L-type VGCCs play a crucial role in the modulation of acute experimental visceral pain in sheep. The important significance of VGCC L-type inhibitors, applied together with opioids in weakness of clinical nociception, have been revealed by Gullapalli and Ramarao [33], that L-type channel modulation by 1,4-dihydropyridines (nimodipine and lercanidipine) potentiates kappa-opioid receptor agonist induced acute analgesia and inhibits the development of tolerance in rats using the tail-flick test. Nimodipine (1 mg·kg−1; *i.p*.) and lercanidipine (0.3 mg·kg−1; *i.p*.) used in this study produced no tail-flick analgesia, but administered that in these doses, 15 min prior analgesic doses of selective kappa-opioid agonists (U 50,488, PD 117,302 and U 69,593) significantly potentiated the analgesia produced by three kappa-opioid receptor agonists. These results strongly suggested a functional role of L-type Ca2+ channels in the regulation of pain sensitivity and mechanism of kappa-opioid analgesia. Last results by Qian et al. [17] suggest also, an important role of VGCCs in rat visceral hypersensitivity by 2,4,6-trinitrobenzenesulfonic acid provoked car nimodipine and SNX-482 prevented it.

In our study, saline and 20% DMSO *i.c.v.* infused during 1 min in volume of 100 μL did not change the ruminal motor activity during 30 min before DD40 and after introduced empty balloon, it was 1.38 ± 0.14 c·min−1. The results obtained were nonsignificant in comparison with the results obtained after the intraduodenal balloon placed and no influencing on the interpretation of the results obtained. Mechanical duodenal distension by balloon 10 cm in length with 40 mL of warm water (DD40) provoked during and after 5 min total inhibition of spiking activity [30, 32] or contraction of the rumen and duodenum during 8–12 min (p ≤ 0.01, **Figure 2**), approximately 85%. These effects, lasting 20 min after DD termination (average 47.3%) were statistically significant (p ≤ 0.05) in comparison to the control values (**Figure 2**). During 5 min episode of DD40, only one to two ruminal contractions were registered in six animals tested. Singular contraction recurred after terminations of DD40 immediately, but their number did not exceed the values of control contractions.

It is known that DD40 provoked stimulation of behavioral signs, clinical symptoms, and statistically significant increase in plasma cortisol and catecholamine concentrations (**Table 1**, **Figures 3**–**6**). *I.C.V.* premedication, by VGCCIs attenuated ruminal motor inhibition by 5 min episode DD, provoked during 30 min average 39.4%, e.g., from 56.8% after DD to 24.2, 18.1 and 9.6% after diltiazem, nifedipine, and verapamil premedication, respectively. The most preventing for the 5 min DD inhibitory influence on ruminal motility was verapamil in comparison to the control values. All the VGCCIs in premedication use inhibited statistically significant behavioral signs, such as looking around, defecation and/or urination, head movements, lying down, and clinical symptoms, such as tachycardia and hyperventilation by 120 min after DD persistent (**Table 1**).

Five-minute DD episode increased the plasma cortisol concentration average for 153% during 120 min after DD, in comparison to the control values. Ten-minute VGCCIs premedication diminished the plasma cortisol release by an average of 139.5 % (diltiazem), and 141% (nifedipine and verapamil) (p ≤ 0.001; **Table 3**, **Figure 3**) respectively.

Five-minute DD episode increased the plasma catecholamine concentration average: E 768%, NE 98.5%, and DA 124% during 120 min after DD. Diltiazem, nifedipine, and verapamil minimized the increase of plasma catecholamine concentration, which was caused by visceral pain, provoked by duodenal distension. Average of catecholamine release inhibition by VGCCIs were for E—773.3%, NA—90%, and DA—90.2%, during 120 after 5 min DD episode (p ≤ 0.001; **Table 4**, **Figures 4**–**6**). The most anticatecholaminergic activity was detected for diltiazem. In our study, we found that all the VGCCIs—whatever their chemical origin—in premedication attenuated vegetative signs and clinical symptoms, HPA and SAS stimulation axes caused by acute 5 min duration nocifensive factor (duodenal distension) in sheep.

This confirms the results obtained by Qian et al. [17] that Cav1.2 and Cav2.3 channels in colonic primary sensory neurons play an important role in visceral inflammatory hyperalgesia, which may be a potential therapeutic target, because L-type and R-type selective colonic channel blockers may block calcium currents which are importantly increased in colonic dorsal root ganglion (DRG) neurons of 2,4,6-trinitrobenzenesulfonic acid-treated rats in comparison with control animals. The author cited above concluded that L-type channel antagonist (nimodipine) and R-type channel antagonist (SNX-482) attenuates visceral pain in 2,4,6-trinitrobenzenesulfonic acid intrathecal injected. The results obtained confirm the hypothesis that L-type and/or R-type calcium channels play a more crucial role in pathology of visceral pain in animals.

A moderate degree of mechanical duodenal distension (DD20 and DD30) in sheep reduced the frequency of forestomach and abomasum motor activity by 45 and 52%, respectively [30], whereas, strong distension (DD40 and DD80) provoked the total contraction inhibition in conscious animals accompanied by the acute visceral pain [21, 33]. There is a direct relationship between the viscero-visceral reflex and visceral pain [34]. Visceral pain is a general sign involved in many gastro-duodenal and gastro-colonic disorders, such as colic, inflammatory processes, and other diseases. These symptoms are accompanied by stimulation of the HPA (neuropeptides, hormones, e.g., cortisol) and SAS (catecholamine, 5-HT, neuropeptides) axes and the exacerbation of motivational and motor CNS structures (limbic system) involved in many quinine, neuropeptides, and necrohormones release useful in alarm reactions and defense of animals.

*I.C.V.* application of VGCCIs in 10 min premedication prevented nocifensive signs of behavior, clinical symptoms, increase plasma cortisol and catecholamine concentration in periphery, and perhaps in CNS structures, as well. The molecular mechanisms of these processes are the result of the L-type voltage-gated calcium channel inhibitors blockage of specific Ca2+ receptors by the drugs tested. Calcium channels receptor blockage by VGCC inhibitors attenuates visceral pain by inhibiting nocifensive neurohormone/neurotransmitters release in CNS and in peripheral nervous system, due to the fact that Ca2+ ions cannot bind to their specific receptor for depolarization of presynaptic neuronal membrane and promote the release of nocifensive substances.

## **4.1. Other types of calcium channel blockers used in the treatment of pain**

animals tested. Singular contraction recurred after terminations of DD40 immediately, but their

It is known that DD40 provoked stimulation of behavioral signs, clinical symptoms, and statistically significant increase in plasma cortisol and catecholamine concentrations (**Table 1**, **Figures 3**–**6**). *I.C.V.* premedication, by VGCCIs attenuated ruminal motor inhibition by 5 min episode DD, provoked during 30 min average 39.4%, e.g., from 56.8% after DD to 24.2, 18.1 and 9.6% after diltiazem, nifedipine, and verapamil premedication, respectively. The most preventing for the 5 min DD inhibitory influence on ruminal motility was verapamil in comparison to the control values. All the VGCCIs in premedication use inhibited statistically significant behavioral signs, such as looking around, defecation and/or urination, head movements, lying down, and clinical symptoms, such as tachycardia and hyperventilation by

Five-minute DD episode increased the plasma cortisol concentration average for 153% during 120 min after DD, in comparison to the control values. Ten-minute VGCCIs premedication diminished the plasma cortisol release by an average of 139.5 % (diltiazem), and 141%

Five-minute DD episode increased the plasma catecholamine concentration average: E 768%, NE 98.5%, and DA 124% during 120 min after DD. Diltiazem, nifedipine, and verapamil minimized the increase of plasma catecholamine concentration, which was caused by visceral pain, provoked by duodenal distension. Average of catecholamine release inhibition by VGCCIs were for E—773.3%, NA—90%, and DA—90.2%, during 120 after 5 min DD episode (p ≤ 0.001; **Table 4**, **Figures 4**–**6**). The most anticatecholaminergic activity was detected for diltiazem. In our study, we found that all the VGCCIs—whatever their chemical origin—in premedication attenuated vegetative signs and clinical symptoms, HPA and SAS stimulation axes caused by acute 5 min duration nocifensive factor (duodenal distension) in sheep.

This confirms the results obtained by Qian et al. [17] that Cav1.2 and Cav2.3 channels in colonic primary sensory neurons play an important role in visceral inflammatory hyperalgesia, which may be a potential therapeutic target, because L-type and R-type selective colonic channel blockers may block calcium currents which are importantly increased in colonic dorsal root ganglion (DRG) neurons of 2,4,6-trinitrobenzenesulfonic acid-treated rats in comparison with control animals. The author cited above concluded that L-type channel antagonist (nimodipine) and R-type channel antagonist (SNX-482) attenuates visceral pain in 2,4,6-trinitrobenzenesulfonic acid intrathecal injected. The results obtained confirm the hypothesis that L-type and/or R-type calcium channels play a more crucial role in pathology of visceral pain in

A moderate degree of mechanical duodenal distension (DD20 and DD30) in sheep reduced the frequency of forestomach and abomasum motor activity by 45 and 52%, respectively [30], whereas, strong distension (DD40 and DD80) provoked the total contraction inhibition in conscious animals accompanied by the acute visceral pain [21, 33]. There is a direct relationship between the viscero-visceral reflex and visceral pain [34]. Visceral pain is a general sign involved in many gastro-duodenal and gastro-colonic disorders, such as colic, inflammatory

(nifedipine and verapamil) (p ≤ 0.001; **Table 3**, **Figure 3**) respectively.

number did not exceed the values of control contractions.

120 min after DD persistent (**Table 1**).

238 Pain Relief - From Analgesics to Alternative Therapies

animals.

Voltage-gated calcium channels are made with subunit α1 which forms a channel pore and subunit α2δ, which facilitates movement to the membrane surface [35]. There are ten different α1δ subtypes and four α2δ. α2δ1 and α2δ2 subtypes bind gabapentin and pregabalin. Subtype 1 exists principally in the intermediate matrix (dentate gyrus, insula, cortex, and amygdala) [36].

Subtypes α2δ1 exhibit expression also in dorsal root ganglia, spinal cord and in the small intestine smooth muscle, together with N-type calcium channels [36]. Subtype 2 is found in the periaqueductal gray matter, spinal cord and as diffused all over CNS, but not in the colon or duodenal smooth muscle [37]. Pregabalin and gabapentin bind with subtypes α2δ in the cytoplasm and prevent calcium channels expression on the plasmatic membranes [38]. Preventing the binding and expression blocks calcium conductance and in consequence substance P (SP), calcitonin gene related peptide (CGRP) and glutamate cannot be released from primary afferent neurons [35, 39]. Prevention of nocifensive neurotrasmitters release by gabapentine and pregabaline occurs only during pathological processes, in which calcium channels are up-regulated and activated [40]. Both pregabaline and gabapentine are central analgesics [39]. Gabapentinoids inhibited visceral hypersensitivity in the experimental animals, as well as irritable bowel syndrome in humans [41]. Smalls doses of gabapentine administered with morphine inhibit *i.p.* acid injection induced writhing syndrome in rats, which were ineffective when the drugs were applied separately.

Gabapentenoids not only inhibit central nociceptive transmission, but also enhance the intestine susceptibility to distension, possibly by blockage of α2δ subtypes in the smooth muscle [42, 43].

Other calcium channels can be also involved in the development of visceral hypersensitivity. Stimulation of T-type calcium channels, subtype Cav3.2 on the primary signaling visceral afferents was associated with symptoms similar to the irritable bowel syndrome in the animal model. Behavioral symptoms resolved after the application of T-type calcium channels inhibitor [44]. Afferent transduction from mesentery in the experimental intestinal ischaemia was blocked by nifedipine, an L-type calcium channels inhibitor [45].
