Selective alkylation of rat urinary bladder muscarinic receptors with 4-DAMP mustard reveals a contractile function for the M2 muscarinic receptor

Abstract

Our previous data indicate that M3 muscarinic receptors mediate carbachol induced bladder contractions. The data presented here were obtained by selective alkylation of M3 receptors with 4-DAMP mustard and suggest that the M2 receptor subtype may be involved in inhibition of beta-adrenergic receptor induced relaxation, therefore, allowing recontraction. Alkylation resulted in 85% of M3 receptors and 65% of M2 receptors unable to bind radioligand as demonstrated by subtype selective immunoprecipitation. Rat bladder strips subjected to our alkylation procedure contracted submaximally, and direct carbachol contractions were inhibited by antagonists with affinities consistent with M3 receptor mediated contraction. In contrast, the affinities of antagonists for inhibition of carbachol induced recontractions following isoproterenol stimulated relaxation in the presence of 90 mM KCl, indicated a contractile function for the M2 receptor that was not observed in control strips. In conclusion, these studies demonstrate a possible role for the M2 subtype in bladder smooth muscle contraction.

Figure 1. Affinity of Anticholinergics for Inhibition of Contraction of Control and Alkylated Rat Bladder Strips in-vitro.

The closed symbols with error bars represent reported affinity ranges for the individual subtypes (15). The open symbols (±S.E.M.) represent the affinities derived from control and alkylated rat bladder strips.

Figure 2. Affinity of Anticholinergics for Inhibition of Contraction of Control and Alkylated Rat Bladder Strips in-vitro in the presence of 90 mM KCl and 30 μM Isoproterenol

The closed symbols with error bars represent reported affinity ranges for the individual subtypes (15). The open symbols (±S.E M.) represent the affinities derived from control and alkylated rat bladder strips

Figure 3. Carbachol concentration-response displacement curves and Schild plot (inserts) for methoctramine’s effect on recontractions of rat bladder strips in which muscarinic receptors were alkylated by 40 nM 4-DAMP mustard in the presence of 30 nM methoctramine in-vitro.

The concentration of methoctramine used is shown in the legend. Each curve represents the average responses of 5–6 muscle strip preparations expressed as the percent of each individual strip’s maximal carbachol response. These maximal responses (average ± S.E.M.) in grams of tension were 2.1 ± 0.18 for control (square); 2.1 ± 0.36, 2.1 ± 0.36 and 2.2 ± 0.38 for 0.3 (circle), 1 (triangle), and 3 μM (upside down triangle) methoctramine respectively. The carbachol EC₅₀ for the antagonist-free strips was 5.1 ± 0.34 μM. The slope of the Schild plot was significantly less than unity.

Figure 4. Precipitation of M₂ and M₃ Muscarinic Receptor Subtypes from Normal Control, Time Control, and 4-DAMP Mustard Treated Rat Bladder Strips

Receptors were labeled with [³H] QNB and solubilized as described in the text. Data shown are average fmoles of receptor/mg solubilized protein ±S.E.M. or the ratio of M₂:M₃ receptors from pooled normal control (clear), time control (shaded) and 4-DAMP mustard treated (striped) bladders Approximately 1 g of tissue per assay was used, n= 3 performed in triplicate for normal control, n= 1 performed in quadruplicate for time control and n= 1 performed in quadruplicate for 4-DAMP mustard treated bladders.