Diltiazem

Before subjecting the segments to different experimental conditions to obtain the vasoreactive measurements described below, the aortic segments were stretched and stabilized to one of the following preloads of 10, 16, 20, 28, 30, 40, 50, or 65 mN in Krebs Ringer solution at 37°C. Aortic segments of the same mouse were stretched to different preloads, and measurements were performed in parallel. Randomization of the segments was employed to exclude non-specific effects of the changes in aortic physiology between the proximal and distal segments of the thoracic aorta.
K⁺ concentration-contraction curves (KCC) were measured at different preloads by gradually increasing external K⁺ from 5.9 to 10, 15, 20, 25, 30, 35, 40, and 50 mM by isosmotic replacement of Na⁺ by K⁺ in Krebs Ringer solution. Concentration-contraction curves for α₁ adrenoceptor stimulation of the aortic segments with phenylephrine (PECC) were cumulatively determined for concentrations of 3 × 10⁻⁹ to 3 × 10⁻⁶ M. Both KCC and PECC were fitted with sigmoidal concentration-response equations with variable slope (GraphPad Prism), which revealed maximal responses (Emax) and (the logarithm of) the concentration resulting in 50% of the maximal contraction (logEC₅₀ for PE and EC₅₀ for K⁺).
Phasic contractions by PE were measured by adding 1 μM PE to the segments incubated for 3 min in Krebs Ringer solution without extracellular Ca²⁺ and with 1 mM EGTA. These contractions are mediated by the IP₃-dependent release of contractile Ca²⁺ from the SR (Fransen et al., 2015 (link)). After the phasic contraction, extracellular Ca²⁺ (3.5 mM) was added to measure the tonic contraction induced by Ca²⁺ influx in the PE-sensitized segment. Next, 35 μM diltiazem was added, to block the Ca²⁺ influx via voltage-gated Ca²⁺ channels. In this way, the contribution of VGCC and NSCC to the tonic contraction can be ascertained (Fransen et al., 2015 (link)). In some experiments, segments were incubated for 5 min with levcromakalim (1 μM), a K_ATP channel agonist, to repolarize aortic segments to the K⁺ equilibrium potential.
To assess the basal release of NO, contractions by elevated K⁺ or PE were measured in separate segments of the same mouse in the absence and presence of 300 μM L-NAME to inhibit eNOS. We have previously shown that this is the most sensitive way to measure basal NO release (van Langen et al., 2012 (link); Leloup et al., 2015b (link)). Relaxation of 1 μM PE-precontracted segments by endogenous and exogenous NO release was measured by constructing concentration-relaxation curves for acetylcholine (ACh, 3 × 10⁻⁹ to 3 × 10⁻⁶ M) and diethylamine NONOate (DEANO, 3 × 10⁻¹⁰ to 3 × 10⁻⁶ M), fitting the curves with sigmoidal concentration-response equations with variable slope (GraphPad Prism) to obtain E_max- and logEC₅₀-values.

Assay of sperm binding to and release from oviduct isthmic cell aggregates was conducted according to the previous report [9 (link)]. Briefly, the spherical oviduct isthmic cell aggregates were washed twice in fresh dmTALP medium. Then, 2 µL spermatozoa at a final concentration of 5 × 10⁵ cells/mL were added into a 50 µL droplet containing 10 oviduct isthmic cell aggregates. Spermatozoa and the oviduct isthmic cell aggregates were incubated at 39 °C for 15 min for sperm binding to form the complexes. Then, the complexes were incubated without or with 1 nM NPPC for 30 min at 39 °C. In some experiments, the complexes were pre-incubated with l-cis-Diltiazem (50 µM), NNC 055-0396 (2 μM), or BAPTA/AM (5 μM) for 5 min at 39 °C, and then incubated without or with NPPC. At the end of incubation, the free and loosely attached spermatozoa were removed from the oviduct isthmic cell aggregates by washing with dmTALP medium. The aggregates were fixed with 4% paraformaldehyde for 10 min and then transferred to a 3 µL droplet in the microscope slide. Images were captured using Axio Vert. A1 microscope (Zeiss, Oberkochen, Germany). The number of spermatozoa bound to the periphery of each aggregate was enumerated, and the circumference of the aggregate was calculated using Labscope V 3.2 software (Zeiss, Oberkochen, Germany). The number of spermatozoa bound per millimeter of circumference was calculated for each aggregate, and the average number of spermatozoa from 10 aggregates was used for statistical analysis. All the cultures were incubated at 39 °C under 5% CO₂.

The capacitated spermatozoa were incubated in dmTALP medium, supplemented with different concentrations of NPPC (0.01–10 nM), NNC 055-0396 (CatSper channel inhibitor, 2 μM), 8-Br-cGMP (1 mM), and/or l-cis-Diltiazem (CNG channels inhibitor, 50 μM) for 30 min. The sperm motion was detected using a CASA system (Version 12 CEROS, Hamilton Thorne Research, Beverly, MA, USA). The parameters included straight-line velocity (VSL), amplitude of lateral head displacement (ALH), average path velocity (VAP), curvilinear velocity (VCL), beat cross frequency (BCF), and percentage of linearity (LIN, VSL/VCL × 100%). For each experimental condition, five random fields were evaluated for a minimum total of 100 cells in each field.