Functional involvement of L-type calcium channels and cyclic nucleotide-dependent pathways in cadmium-induced myometrial relaxation in rats

Abstract

Modulation of myometrial spontaneity by cadmium (Cd) and its regulatory pathways was studied in rat uterus in the absence and presence of blockers of different signaling pathways. Isometric tension in myometrial strips, under a resting tension of 1 g, mounted in organ bath containing Ringer–Locke solution (RLS) continuously aerated with carbogen, was measured using data acquisition system-based physiograph and Lab Chart Pro V7.3.7 software. Mean integral tension was measured for 8 min. Cd (1 nM–0.1 mM) not only produced concentration-dependent inhibitory effect on rat myometrium but it (10 µM) also significantly (p < 0.05) inhibited calcium chloride and BAY K-8644-induced myometrial contraction. Glybenclamide (10 µM), 4-aminopyridine (1 mM), and propranolol (10 µM) failed to significantly attenuate Cd-induced inhibitory responses, while L-NAME (0.1 mM), 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 25 µM), and 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22536; 1 µM) significantly (p < 0.05) produced inhibitory effects on Cd-induced myometrial relaxation. Phenylephrine (1 nM–10 µM) and salbutamol (0.01 nM–0.1 µM)-induced relaxant effects on rat myometrium were significantly potentiated by 10 µM Cd. Thus based on the results of present functional study, it may be inferred that inhibitory effects of Cd on rat myometrium are mediated through blockade of L-type calcium channels and activation of NOS-NO-sGC and/or AC-cAMP pathways.

Keywords

Cadmium L-type calcium channel adenylyl cyclase guanylyl cyclase rat myometrium

Introduction

Cadmium (Cd) is one of the most abundant and potent hazardous metallic environmental pollutant and the geometric blood level of Cd in general population of ≥1 year of age is 0.315 µg/L.¹ Exposure to Cd occurs primarily through ingestion of contaminated water, food, and to a significant extent through inhalation and cigarette smoking.² Apart from cancer, immune, respiratory, and cardiovascular disorders,² Cd results in reproductive defects like subfertility or abortions in humans³ and animals.⁴ Cd accumulates in human endometrial tissue and its high levels have been reported in females with the history of smoking.⁵ Smoking during pregnancy increases the contractile sensitivity and activity of the myometrium to oxytocin by upregulating oxytocin receptor mRNA and increases the risk of premature delivery in cigarette-smoke-exposed Wistar rats.⁶ Cd inhibits angiogenesis in endometrial cells which results in endometrial dysfunctions and increased risk for fertility problems.⁷

Cd produces inhibitory effect on human myometrium spontaneity while increases and decreases the response of Ca²⁺ and oxytocin at lower (1 nM–10 nM) and higher concentrations, respectively,⁸ and inhibitory effect on bovine myometrium.⁹ On guinea pig ileum, it has been reported to produce both the inhibitory and excitatory effects.^10,11 But its dose-dependent differential effects, that is, stimulatory at lower concentration and inhibitory at higher concentration have been observed on rat thoracic aorta.¹² Cd contracts ileal longitudinal muscle through release of cholinergic transmitter from parasympathetic-nerve terminals, which depends on external Ca²⁺ apart from smaller hyoscine-resistant contractile effect, presumably due to direct action on smooth muscle cells.¹¹ Chronic Cd intoxication at 5 mg/kg body weight resulted in an increase in norepinephrine response in rat mesenteric arteries¹³ while endothelin, noradrenaline, and angiotensin II achieved lower maximal contraction in Cd-induced hypertensive rat aorta as compared to normotensive rats.¹⁴ But in vitro Cd (10 µM) treatment for 24 h augmented α₁-adrenoceptor-mediated contractile response in rat aorta even though endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) were upregulated by Cd.¹⁵ Takahashi et al.¹⁵ also observed increase of nitric oxide (NO)-induced and β-adrenoceptor-mediated relaxing responses by Cd treatment in rat aorta. Thus, the regulatory mechanism(s) of Cd seem to vary not only with concentration but also the type of smooth muscles (myometrium, ileum, aorta, mesenteric artery) and between species, namely human, bovine, guinea pigs, and rats.

Ion transport and signal transduction, which include intracellular mobilization of second messengers such as inositol triphosphate,¹⁶ inhibition of plasma membrane calcium channels,¹⁷ and inhibition of plasma membrane Ca²⁺-ATPase,¹⁸ have been identified as the cellular targets of Cd in different smooth muscles. Apparently, there is paucity of detailed information on effect of Cd on myometrial activity, its cellular targets and mechanistic pathways, including cyclic nucleotide-dependent pathways. Hence the present study was undertaken to study the direct effect of Cd and its mechanistic pathways on isolated myometrial strips of rats.

Materials and methods

Chemicals

Cadmium chloride (CdCl₂) monohydrate and prostaglandin F₂ alpha (PGF₂α, lutalyse) were purchased from Himedia (Mumbai, Maharashtra, India) and Pfizer (Mumbai, Maharashtra, India), respectively. Glybenclamide—a specific K_ATP channel blocker, 4-aminopyridine -a specific K_v channel blocker, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl] phenyl) pyridine-3-carboxylic acid methyl ester (BAY K-8644)—a specific L-type calcium channel agonist, Nω-nitro-l-arginine methyl ester hydrochloride (L-NAME)—a NOS inhibitor, 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ)—a specific blocker of sGC, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22536)—a specific blocker of adenylyl cyclase, phenylephrine hydrochloride—an α₁ adrenoceptor agonist, salbutamol—a β₂ adrenoceptor agonist, and propranolol—a nonspecific β-adrenoceptor blocker were procured from Sigma–Aldrich (St. Louis, Missouri, USA). Propranolol was dissolved in ethanol and 4-aminopyridine, BAY K-8644, ODQ, and glybenclamide were dissolved in dimethyl sulfoxide while CdCl₂, PGF₂α, L-NAME, SQ 22536, phenylephrine, and salbutamol were dissolved in triple distilled water and stored at 4°C but L-NAME and SQ 22536 solutions were stored at −20°C. Further dilutions of the required concentration were made in freshly prepared Ringer–Locke solution (RLS) on the day of use. The solvents used for preparation of chemical solution(s) had no effect on the response of tissue at the concentration(s) used.

Experimental animals

Study was conducted on female albino rats (Rattus norvegicus; strain Wistar) weighing 150–180 g procured from Laboratory Animal Resource Section, Indian Veterinary Research Institute (Izatnagar, Uttar Pradesh, India), and maintained under standard managemental conditions with 12-h dark and 12-h light cycle. An acclimatization period of 15 days was ensured before start of the experiment, to acclimatize the animals to the new environment and also switch over to new diet. Experimental studies were undertaken after approval of the Institutional Animal Ethics Committee, DUVASU, Mathura (approval no. IAEC/DUVASU/20/12 dated 1 December 2012) as per Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines.

Selection of animals

Di-estrus stage cyclic rats were selected based on vaginal smear examination¹⁹ and used for experimentation as rat myometrium in di-estrus stage exhibited regular and consistent amplitude, frequency, and mean integral tension (MIT) along with longevity of isolated tissue apart from short stabilization time compared to the myometrium from estrogenized rats. For each experimental protocol, eight to ten animals were used.

Collection of tissue

Cyclic adult female rats during di-estrus stage were ethically sacrificed as per the method described by Ghosh²⁰ after anesthetizing with pentobarbitone sodium (40 mg/kg body wt i.p.). After decapitation, mid-ventral incision was made and both the uterine horns were dissected out and carefully placed in a petri dish containing aerated RLS having NaCl-154 mM/L; KCl-5.6 mM/L; calcium chloride (CaCl₂), 2H₂O-2.2 mM/L; NaHCO₃-6.0 mM/L; d-glucose-5.5 mM/L; and pH of 7.4. Tissues were cleaned by removing connective and adipose tissues without any damage to uterine horn.

Mounting of tissue and recording of isometric tension

Myometrial strips were mounted in thermostatically controlled (37.0 ± 0.5°C) organ bath (Ugo Basile, Italy) of 10 ml capacity containing RLS, continuously aerated with carbogen (95% O₂ + 5% CO₂) under a resting tension of 1 g. During the equilibration period of 30 min, physiological salt solution in tissue bath was changed almost every 10 min. Isometric tension in tissue was measured by force transducer (AD Instruments, Australia) and recorded with the help of data acquisition system-based physiograph using Lab Chart Pro V7.3.7 software (Powerlab, AD Instruments; Australia). Contact period of Cd with isolated myometrial strips in the tissue bath was 8 min. Tissues were exposed to different concentrations of Cd (10⁻⁹ to 10⁻⁴ M). Myometrial strips were incubated with different antagonists/blockers for a period of 30 min.

Data analysis

Concentration–response curves were constructed by measuring the MIT for 8 min using Labchart pro V 7.3.7 software as per the formula given by Aaronson et al.²¹ Maximum contraction (E_max)/maximum relaxation (R_max) and median effective dose (EC₅₀) values were determined by nonlinear regression analysis using Graph Pad Prism 4.0 (Graph Pad, La Jolla, San Diego, USA) and pD₂ value (potency) was calculated as −log of EC₅₀. Results are expressed as mean ± SEM. Multiple mean values were analyzed using two-way ANOVA followed by Bonferroni post-hoc test or one-way ANOVA followed by Tukey’s post-hoc test, while two mean values were analyzed by student’s t test to compare the difference between two different treatments.

Results

Selection of di-estrus stage rat myometrium

Compared to the initial stabilization time, longevity of tissue responsiveness and spontaneity features (amplitude, frequency, and MIT) in isolated myometrial strips mounted in RLS from estrogenized rats, myometrial strips from normally cyclic rats during di-estrus stage were found to possess significantly (p < 0.05) higher amplitude, MIT and longevity, and significantly lower stabilization period as evident from the data summarized in Table 1 and representative physiographic recordings (Figure 1). Therefore, the study was undertaken on isolated myometrial strips of di-estrus stage cyclic rats.

Table 1.

Comparative amplitude, frequency, mean integral tension, stabilization time, and longevity of myometrial strips from estrogenized and cyclic rats during di-estrus stage.

Stage	Amplitude (g)	Frequency (bpm)	MIT (g)	Stabilization time (h)	Longevity of tissue (h)
Estrus (n = 16)	0.95 ± 0.07	2.94 ± 0.16	0.30 ± 0.01	0.77 ± 0.12	4.5 ± 0.14
Di-estrus (n = 16)	1.24 ± 0.10^b	3.08 ± 0.43	0.48 ± 0.03^b	0.36 ± 0.01^b	5.10 ± 0.07^b

^aVertical bars represent SEM. Data were analyzed by student’s t test followed by unpaired t tests.

^bp < 0.05 vs. estrus.

Figure 1.

Representative physiograph recordings showing normal spontaneity pattern of isolated rat myometrial strips from di-estrus (a) and estrus (b) stage rats, mounted in RLS. RLS: Ringer–Locke solution.

Effect of cadmium on myometrial spontaneity

Following equilibration, rat myometrial strips (n = 16) exhibited consistent and regular pattern of myogenic spontaneity with an average amplitude, frequency, and MIT of 1.24 ± 0.10 g; 3.08 ± 0.43 beats per minute (bpm) and 0.48 ± 0.03 g, respectively. CdCl₂ (1 nM–0.1 mM, at 1 log dose unit) produced concentration-dependent inhibitory effect (Figure 2(a)) on rat myometrium. Figure 2(b) illustrates the cumulative dose–response curve (DRC) of CdCl₂ on rat myometrium. The pD₂ and R_max values of CdCl₂ were calculated to be 5.15 ± 0.15 (n = 8) and 113.30 ± 5.68% (n = 8), respectively.

Figure 2.

Representative physiograph recording (a) and cumulative concentration response curve (b) of cadmium chloride (1 nM to 0.1 mM) on rat myometrium. Vertical bars represent SEM (n = 8).

Effect of cadmium chloride on CaCl₂-induced myometrial contraction in Ca²⁺-free high K⁺ (80 mM) depolarizing solution

To assess whether Cd directly reduces calcium entry through L-type of calcium channels, effect of cumulative doses of CaCl₂ (1 µM–10 mM) was recorded in Ca²⁺-free high K⁺ (80 mM) depolarizing solution alone and in the presence of 10 μM Cd as this dose produced submaximal (∼80%) relaxation in rat myometrium. In this protocol, myometrial strips were initially exposed to Ca²⁺-free RLS containing 3 mM ethylene glycol tetraacetic acid and after two successive washings with this solution at 4-min interval, tissues were incubated with Ca²⁺-free high K⁺ depolarizing solution (to open the L-type Ca²⁺ channels) before recording the dose response of CaCl₂ alone and in the presence of Cd (10 µM). In the presence of CdCl₂, the DRC of CaCl₂ was significantly (p < 0.05) shifted towards right (Figure 3(a)) with significant (p < 0.05) reduction of potency of CaCl₂ (2.47 ± 0.39, n = 7 vs 3.89 ± 0.24, n = 7) without significant decrease in efficacy (0.70 ± 0.05 g, n = 7 vs 0.79 ± 0.10 g, n = 7).

Figure 3.

Cumulative dose–response curve of CaCl₂ in Ca²⁺-free high K⁺ (80 mM) depolarizing solution (a; n = 7) and that of BAY K-8644 (b; n = 6) on rat myometruim in the presence of CdCl₂ (10 µM). Vertical bars represent SEM. Data were analyzed by two-way ANOVA followed by Bonferroni post-hoc tests. *p < 0.05. CaCl₂: calcium chloride; ANOVA: analysis of variance.

Effect of cadmium on BAY K-8644-induced myometrial contraction

To further strengthen the finding of reduced calcium entry through L-type calcium channel by Cd in rat myometrium, cumulative dose response of BAY K-8644 (0.1 nM–10 µM) alone and in the presence of Cd (10 µM) were recorded. As shown in Figure 3(b), BAY K-8644 produced dose-dependent contractile effect on rat myometrium which was significantly (p < 0.05) attenuated by Cd (−0.30 ± 0.06 g, n = 6) compared to that of BAY K-8644 alone (0.51 ± 0.08 g, n = 6) but without any significant change in potency (6.61 ± 0.29, n = 6 vs 5.62 ± 0.45, n = 6).

Effect of L-NAME on cadmium-induced myometrium relaxation

To investigate the possible involvement of NO in CdCl₂ (1 nM–0.1 mM)-induced relaxation, effect of CdCl₂ was studied on rat myometrium in the presence of L-NAME. In the presence of L-NAME (0.1 mM), the DRC of CdCl₂ was significantly (p < 0.05) shifted toward right (Figure 4(a)) but without any significant decrease in potency and efficacy of CdCl₂. The pD₂ and R_max values of CdCl₂ alone and in the presence of L-NAME were found to be 5.13 ± 0.24 (n = 7) vs 4.70 ± 0.54 (n = 7) and 116.73 ± 5.23% (n = 7) vs 100.52 ± 6.44% (n = 7), respectively.

Figure 4.

Cumulative concentration response curves of cadmium chloride alone and in the presence of 0.1 mM L-NAME (a) and 25 µM ODQ (b) on isolated rat myometrial strips (n = 7). Vertical bars represent SEM. Data were analyzed by two-way ANOVA followed by Bonferroni post-hoc tests. *p < 0.05. L-NAME: Nω-nitro-l-arginine methyl ester hydrochloride; ODQ: 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one.

Effect of ODQ on cadmium-induced myometrium relaxation

To study the involvement of NOS-NO-sGC pathway in mediating CdCl₂-induced myometrial relaxation, cumulative dose response of CdCl₂ alone and CdCl₂ in the presence of ODQ (25 µM) was recorded. In the presence of ODQ, the DRC of CdCl₂ was significantly (p < 0.05) shifted toward right (Figure 4(b)) with significant (p < 0.05) inhibition in efficacy but nonsignificant reduction of potency. The pD₂ and R_max values of CdCl₂ alone and in the presence of ODQ were found to be 5.18 ± 0.16 (n = 7) vs 4.66 ± 0.19 (n = 7) and 116.73 ± 5.23% (n = 7) vs 95.69 ± 7.30% (n = 7), respectively.

Effect of glybenclamide on cadmium-induced myometrium relaxation

To elucidate the possible involvement of adenosine triphosphate-sensitive potassium channels (K_ATP) in CdCl₂-induced rat myometrial relaxation, effect of CdCl₂ was studied in the presence of glybenclamide (10 µM). Neither the DRC of CdCl₂ was shifted either way by glybenclamide as shown in Figure 5 nor was there any significant decrease in potency and efficacy of CdCl₂. The pD₂ value of CdCl₂ alone and in the presence of glybenclamide were almost comparable and found to be 4.99 ± 0.17 (n = 6) vs 4.89 ± 0.19 (n = 6) but R_max value reduced by almost 15% as the R_max values were found to be 96.53 ± 3.26% (n = 6) and 113.71 ± 7.15% (n = 6) in the presence and absence of glybenclamide, respectively.

Figure 5.

Effect of glybenclamide (10 µM) and 4-aminopyridine (1 mM) on cumulative concentration response curves of cadmium chloride on isolated rat myometrial strips (n = 6). Vertical bars represent SEM. Data were analyzed by two-way ANOVA followed by Bonferroni post-hoc tests. ANOVA: analysis of variance.

Effect of 4-aminopyridine on cadmium-induced myometrium relaxation

To study the possible involvement of voltage-gated potassium channels (K_v) in CdCl₂-induced rat myometrial relaxation, effect of CdCl₂ alone and in the presence of 4-amonopyridine (1 mM) was studied. In the presence of 4-amonopyridine, the DRC of CdCl₂ was nonsignificantly shifted toward right (Figure 5) and there was no significant (p < 0.05) alteration in potency as the pD₂ values of CdCl₂ alone and in the presence of 4-amonopyridine were found to be 4.99 ± 0.17 (n = 6) vs 5.30 ± 0.17 (n = 6). But R_max value was found to be reduced by almost 19% as R_max values were found to be 113.71 ± 7.15% (n = 6) and 91.63 ± 9.46% (n = 6), respectively, in the absence and presence of 4-aminopyridine.

Effect of cadmium on phenylephrine-induced myometrial relaxation

Effect of cumulative-doses of phenylephrine alone (1 nM–10 µM) and in the presence 10 µM CdCl₂ was studied in PGF₂α (10 µM)-precontracted myometrial strips as this concentration of PGF₂α (10 µM) was found to produce maximal tension on rat myometrium. Phenylephrine produced dose-dependent relaxant effect on rat myometrium and the DRC of phenylephrine was significantly (p < 0.05) shifted toward left (Figure 6(a)) in the presence of CdCl₂ (10 µM) with significant (p < 0.05) increase in potency and nonsignificant increase in efficacy (R_max). The pD₂ values of phenylephrine were found to be 6.22 ± 0.11 (n = 9) and 6.65 ± 0.17 (n = 9) and R_max values were 140.02 ± 14.33% (n = 9) and 189.52 ± 22.03% (n = 9), respectively, in the absence and presence of CdCl₂ (10 µM).

Figure 6.

Cumulative concentration response curves of phenylephrine (a, n = 9) and salbutamol (b, n = 6) alone and in the presence of 10 µM CdCl₂. (c) depicts the cumulative concentration response curves of CdCl₂ alone and in the presence of propranolol (10 µM) or SQ 22536 (1 µM) on isolated rat myometrial strips (n = 6).Vertical bars represent SEM. Data were analyzed by two-way ANOVA followed by Bonferroni post-hoc tests.*p < 0.05. ANOVA: analysis of variance; CdCl₂: cadmium chloride.

Effect of cadmium on salbutamol-induced myometrial relaxation

In the presence of CdCl₂ in PGF₂α (10 µM)-precontracted tissue, salbutamol produced dose-dependent relaxant effect similar to that by phenylephrine and the DRC of salbutamol was significantly (p < 0.05) shifted toward left (Figure 6(b)) with significant (p < 0.05) increase in potency but nonsignificant increase (p < 0.05) in efficacy (R_max). The pD₂ values of salbutamol were found to be 8.13 ± 0.12 (n = 6) and 8.72 ± 0.14 (n = 6), and R_max values were 112.11 ± 11.25% (n = 6) and 150.36 ± 17.06% (n = 6), respectively, in the absence and presence of CdCl₂ (10 µM).

Effect of propranolol on cadmium-induced relaxation of rat myometrium

In order to elucidate possible involvement of adrenergic receptors, especially β-adrenergic receptors, in mediating CdCl₂-induced myometrial relaxation, effect of CdCl₂ alone and in the presence of propranolol (10 µM) was studied. Although the DRC of CdCl₂ was shifted toward right in the presence of propranolol (10 µM) (Figure 6(c)), neither the shift nor the decrease in potency and efficacy of CdCl₂ were statistically significant. The pD₂ values of CdCl₂ alone and in the presence of propranolol were found to be 4.99 ± 0.17 (n = 6) vs 5.16 ± 0.12 (n = 6) while R_max values were 113.71 ± 7.15% (n = 6) vs 104.23 ± 3.52% (n = 6).

Effect of SQ 22536 on cadmium-induced relaxation of rat myometrium

In view of the significant potentiation of phenylephrine- and salbutamol-induced myometrial relaxation by CdCl₂ and failure of propranolol (10 µM) to attenuate CdCl₂-induced relaxation, possible involvement of adrenoceptor-independent adenylyl cyclase–cAMP pathway was studied by observing the effect of cadmium alone and in the presence of SQ 22536 (1 µM). In the presence of SQ 22536, the DRC of CdCl₂ was significantly (p < 0.05) shifted towards right (Figure 6(c)) with nonsignificant (p < 0.05) decrease in both the potency and efficacy of CdCl₂. The pD₂ values of CdCl₂ were found to be 4.99 ± 0.17 (n = 6) and 4.67 ± 0.18 (n = 6) and R_max values were 113.71 ± 7.15% (n = 6) and 87.82 ± 7.43% (n = 6), respectively, in the absence and presence of SQ 22536.

Discussion

Studies at cellular level under in vitro conditions have suggested that Cd exhibits multifarious actions but the precise mechanism(s) are yet to be comprehensively explained.² Our observation of inhibitory effect of Cd on rat myometrium is in agreement with similar findings on both human and bovine myometrium.^8,9 Cd has been reported to produce dose-dependent differential effects on rat thoracic aorta, that is, stimulatory at lower concentration (1 nM–0.1 µM) and inhibitory at higher concentration (1 µM–0.1 mM),¹² but no such effect was observed on rat myometrium. Thus, possible involvement of different mechanistic pathways in Cd-induced actions on different smooth muscles from different species cannot be ruled out.

Calcium is the major regulatory ion in reactivity of most smooth muscles including myometrium. Results of our study suggest that Cd decreases influx of Ca²⁺ through L-type of calcium channels by blocking voltage-dependent calcium channel (VDCC) as it significantly (p < 0.05) shifted the DRC of CaCl₂ toward right with significant reduction in potency (2.47 ± 0.39) in Ca²⁺-free high K⁺ (80 mM) depolarizing solution. Blockade of VDCC is further substantiated by our observation of the significant (p < 0.05) reduction in BAY K-8644-induced myometrial contractions in the presence of submaximal concentration of Cd (10 µM). Similar findings have been reported in human uterine vessels where Cd blocked calcium channel.²² Cd inhibits Ca²⁺ availability in rat aorta¹² and produced dose-dependent inhibitory effect on Ca²⁺-induced contractions in isolated cerebral and peripheral arteries of dog at higher concentration.²³ Cd enters through L-type calcium channel in pituitary cell line (GH₄C₁)²⁴ and neurons²⁵ and acts as a competitive inhibitor of calcium or decreases Ca²⁺ influx; therefore, similar effect of Cd on rat myometrium cannot be ruled out.

NO is known to be involved in a variety of physiological functions, including neurotransmission, vasodilation, and immunity.^26,27 Several reports indicate that nitric oxide synhase III (eNOS), but not NOS II (iNOS), is expressed in nonpregnant rat uterus, whereas both NOS II and III are expressed in pregnant rat uterus.^28,29 In our study, L-NAME, a NOS inhibitor, significantly inhibited Cd-induced inhibitory effect on myometrium which suggests possible stimulation of NOS by Cd. Significant shift of the DRC of Cd towards right with significant decrease in R_max value of Cd in the presence of ODQ (25 µM), a specific inhibitor of guanylyl cyclase, added credence to involvement of NOS-NO-sGC pathway in mediating Cd-induced inhibitory effect on rat myometrium, similar to that reported in rat aorta¹⁵ with enhanced expression of eNOS and iNOS in Cd-treated rats. But on the contrary, Cd has been reported to inhibit NO production in endothelial cells by blocking eNOS phosphorylation³⁰ and increase vasoconstrictor activity of phenylephrine by reducing NO bioavailability by increasing oxidative stress following Cd exposure.³¹

To date, several types of K⁺ channels have been identified in myometrium which include ATP-sensitive K⁺ channel (K_ATP), shaker-like voltage-gated potassium channels (K_V), calcium-activated large conductance potassium channels (BK_Ca), and small conductance calcium-sensitive potassium channels (SK_Ca).³² Voltage-gated K⁺ (K_v) channels control excitability in nonpregnant and pregnant rat myometrium.²¹ In the present study, 4-aminopyridine (1 mM), a specific blocker of K_v channel,³³ and glybenclamide (10 µM), a selective K_ATP channel blocker,³⁴ failed to attenuate inhibitory effect of Cd on rat myometrium, thus suggesting that possibly activation of voltage-dependent and ATP-dependent K⁺ channels do not have any role in CdCl₂-induced myometrial relaxation. Our findings are in confirmation with the observations in bovine mesenteric artery where Cd has been reported not to affect the Ca²⁺-insensitive K⁺ channels.³⁵

Isolated rat uterus throughout natural estrous cycle possesses predominantly beta-adrenergic receptors.³⁶ High-density α₁ and β₂ adrenergic receptors are expressed in the circular and longitudinal layers of rat myometrium, respectively.³⁷ Phenylephrine and salbutamol, respectively selective α₁ and β₂ adrenoceptor agonists, produced concentration-dependent relaxant effect on rat myometrium. Mhaouty-Kodja et al.³⁸ have also reported relaxant effect of phenylephrine on myometrial strips of late pregnant and parturient mouse due to stimulation of β-adrenoceptors at higher concentration of phenylephrine. Significant leftward shift of the DRCs of phenylephrine and salbutamol in the presence of CdCl₂ with significant increase in potency (pD₂) without change in efficacy (R_max) suggests that CdCl₂ possibly enhances relaxant effect of β-adrenoceptor stimulants by affecting some downstream signaling mechanism rather than adrenergic receptors. This hypothesis is supported by our observation that SQ 22536, an inhibitor of adenylyl cyclase, significantly attenuated the CdCl₂-induced tocolytic effect on rat myometrium, whereas propranolol, an antagonist of β- adrenoceptors, failed to produce any significant inhibitory effect on CdCl₂-induced myometrial relaxation.

Summing up our findings, it may be inferred that Cd-produced relaxant effect on rat myometrium and its direct effect and potentiation of adrenoceptors agonists-induced relaxation in rat myometrium is mediated through blockade of calcium entry through L-type calcium channel and/or stimulation of NOS-NO-sGC and AC-cAMP pathways.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Indian Council of Agricultural Research (ICAR), New Delhi, India under Niche Area of Excellence programme to Department of Veterinary Pharmacology and Toxicology, DUVASU, Mathura, India (Grant No.:10(10)/2012-EPD, dated: 23rd March, 2012).

References

ASTDR. ToxGuide^TM for Cadmium, CAS# 7440-43-9. U.S. Department of Health and Human Services Public Health Service, Division of Toxicology and Human Health Sciences, Environmental Toxicology Branch, Atlanta, GA 30333 USA, 2012, p. 1. Available at: www.atsdr.cdc.gov/toxguides/toxguide-5.pdf

Sarkar

Ravindran

Krishnamurthy

. A brief review on the effect of cadmium toxicity: from cellular to organ level. Int J Biotechnol Res 2013; 3: 17–36.

Pollack

Sjaarda

Ahrens

. Association of cadmium, lead and mercury with paraoxonase 1 activity in women. PLoS One 2014; 9: e92152.

Samuel

Stanley

Princess

. Gestational cadmium exposure-induced ovotoxicity delays puberty through oxidative stress and impaired steroid hormone levels. J Med Toxicol 2011; 7: 195–204.

Rzymski

Tomczyk

. Metal status in human endometrium: relation to cigarette smoking and histological lesions. Environ Res 2014; 132: 328–333.

Egawa

Yasuda

Nakajima

. Smoking enhances oxytocin-induced rhythmic myometrial contraction. Biol Reprod 2003; 68: 2274–2280.

Helmestam

Stavreus-Evers

Olovsson

. Cadmium chloride alters mRNA levels of angiogenesis related genes in primary human endometrial endothelial cells grown in vitro. Reprod Toxicol 2010; 30: 370–376.

Sipowicz

Kostrzewska

Laudanski

. Effects of cadmium on myometrial activity of the non-pregnant human. Interactions with calcium and oxytocin. Acta Obstet Gynecol Scand 1995; 74: 93–96.

Yildirim

Macun

. The effects of cadmium, copper and lead on in vitro bovine uterine contractility. Kafkas Univ Vet Fak Derg 2013; 19: 793–799.

10.

Schnieden

Small

. Spasmolytic effects of cadmium and zinc ions upon the guinea-pig isolated ileum preparation. Br J Pharmacol 1971; 41: 488–499.

11.

Asai

Nishimura

Satoh

. Mechanism of cadmium-induced contraction in ileal longitudinal muscle of guinea-pig. Br J Pharmacol 1982; 75: 561–567.

12.

Niwa

Suzuki

. Effects of cadmium on the tension of isolated rat aorta (a possible mechanism for cadmium-induced hypertension). J Toxicol Sci 1982; 7: 51–60.

13.

Skoczynska

Wróbel

Andrzejak

. Lead-cadmium interaction effect on the responsiveness of rat mesenteric vessels to norepinephrine and angiotensin II. Toxicology 2001; 162: 157–170.

14.

Ozdem

Ogutman

. The effects of short-term nifedipine treatment on responsiveness of aortic rings of cadmium hypertensive rats. Clin Exp Hypertens 1999; 21: 423–440.

15.

Takahashi

Poteser

Masui

. Effects of cadmium in vitro on contractile and relaxant responses of isolated rat aortas. Environ Health Prev Med 2004; 9: 251–256.

16.

Smith

Dwyer

Smith

. Cadmium evokes inositol polyphosphate formation and calcium mobilization. Evidence for a cell surface receptor that cadmium stimulates and zinc antagonizes. J Biol Chem 1989; 264: 7115–7118.

17.

Huang

Quayle

Worley

. External cadmium and internal calcium block of single calcium channels in smooth muscle cells from rabbit mesenteric artery. Biophys J 1989; 56: 1023–1028.

18.

Da Costa

Botana

Piñero

. Cadmium inhibits motility, activities of plasma membrane Ca²⁺-ATPase and axonemal dynein-ATPase of human spermatozoa. Andrologia 2015; 48(4): 464–469.

19.

Marcondes

Bianchi

Tanno

. Determination of the estrous cycle phases of rats: some helpful considerations. Braz J Biol 2002; 62: 609–614.

20.

Ghosh

. Anaesthetics used in laboratory animals, In Fundamentals of Experimental Pharmacology. 4th ed. Kolkata: Hilton & Company, 2008, pp. 19–23.

21.

Aaronson

Sarwar

Gin

. A role for voltage-gated, but not Ca²⁺-activated K⁺ channels in regulating spontaneous contractile activity in myometrium from virgin and pregnant rats. Br J Pharmacol 2006; 147: 815–824.

22.

Kostrzewska

Laudański

Bergelin

. Influence of cadmium ions on the reactivity of isolated human uterine arteries. J Toxicol Environ Health 1991; 34: 187–195.

23.

Hayashi

Toda

. Inhibition by Cd²⁺, verapamil and papaverine of Ca²⁺-induced contractions in isolated cerebral and peripheral arteries of the dog. Br J Pharmacol 1977; 60: 35–43.

24.

Hinkle

Kinsella

Osterhoudt

. Cadmium uptake and toxicity via voltage sensitive calcium channels. J Biol Chem 1987; 262: 16333–16337.

25.

Sadiq

Ghazala

Chowdhury

. Metal toxicity at the synapse: presynaptic, postsynaptic and long-term effects. J Toxicol 2012; 2012: 13 2671.

26.

Moncada

Palmer

Higgs

. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Rev 1991; 43: 109–142.

27.

MacMicking

Xie

Q W

Nathan

. Nitric oxide and macrophage function. Annu Rev Immunol 1997; 15: 323–350.

28.

Dong

Fang

Gangula

. Regulation of inducible nitric oxide synthase messenger ribonucleic acid expression in pregnant rat uterus. Biol Reprod 1998; 59: 933–940.

29.

Yallampalli

Dong

Gangula

. Role and regulation of nitric oxide in the uterus during pregnancy and parturition. J Soc Gynecol Invest 1998; 5: 58–67.

30.

Majumder

Muley

Kolluru

. Cadmium reduces nitric oxide production by impairing phosphorylation of endothelial nitric oxide synthase. Biochem Cell Biol 2008; 86: 1–10.

31.

Angeli

Cruz Pereira

de Oliveira Faria

. Cadmium exposure induces vascular injury due to endothelial oxidative stress: the role of local angiotensin II and COX-2. Free Radic Biol Med 2013; 65: 838–848.

32.

Brainard

Korovkina

England

. Potassium channels and uterine function. Semin Cell Dev Biol 2007; 18: 332–339.

33.

Smith

McClur

Smith

. The role of voltage-gated potassium channels in the regulation of mouse uterine contractility. Reprod Biol Endocrinol 2007; 5: 41.

34.

Mandi

Sarkar

Mishra

. Effects of calcium channel blocker, mibefradil and potassium channel opener, pinacidil, on the contractile response of mid-pregnant goat myometrium. Indian J Exp Biol 2005; 43: 795–801.

35.

Stockand

Sultan

Molony

. Interactions of cadmium and nickel with K channels of vascular smooth muscle. Toxicol Appl Pharmacol 1993; 121: 30–35.

36.

Boyle

Digges

. Responses to catecholamines of the rat isolated uterus throughout the natural oestrous cycle. Naunyn Schmiedebergs Arch Pharmacol 1982; 321: 56–62.

37.

Legrand

Vivat

Rigolot

. Selective distribution of alpha-1 and beta adrenoceptors in pregnant rat uterus visualized by autoradiography. J Pharmacol Exp Ther 1991; 256: 767–772.

38.

Mhaouty-Kodja

Houdeau

Cohen-Tannoudji

. Catecholamines are not linked to myometrial phospholipase C and uterine contraction in late pregnant and parturient mouse. J Physiol 2001; 536: 123–131.

Functional involvement of L-type calcium channels and cyclic nucleotide-dependent pathways in cadmium-induced myometrial relaxation in rats

Abstract

Keywords

Introduction

Materials and methods

Chemicals

Experimental animals

Selection of animals

Collection of tissue

Mounting of tissue and recording of isometric tension

Data analysis

Results

Selection of di-estrus stage rat myometrium

Effect of cadmium on myometrial spontaneity

Effect of cadmium chloride on CaCl2-induced myometrial contraction in Ca2+-free high K+ (80 mM) depolarizing solution

Effect of cadmium on BAY K-8644-induced myometrial contraction

Effect of L-NAME on cadmium-induced myometrium relaxation

Effect of ODQ on cadmium-induced myometrium relaxation

Effect of glybenclamide on cadmium-induced myometrium relaxation

Effect of 4-aminopyridine on cadmium-induced myometrium relaxation

Effect of cadmium on phenylephrine-induced myometrial relaxation

Effect of cadmium on salbutamol-induced myometrial relaxation

Effect of propranolol on cadmium-induced relaxation of rat myometrium

Effect of SQ 22536 on cadmium-induced relaxation of rat myometrium

Discussion

Footnotes

Declaration of Conflicting Interests

Funding

References

Effect of cadmium chloride on CaCl₂-induced myometrial contraction in Ca²⁺-free high K⁺ (80 mM) depolarizing solution