Probenecid: novel use as a non-injurious positive inotrope acting via cardiac TRPV2 stimulation

Abstract

Probenecid is a highly lipid soluble benzoic acid derivative originally used to increase serum antibiotic concentrations. It was later discovered to have uricosuric effects and was FDA approved for gout therapy. It has recently been found to be a potent agonist of transient receptor potential vanilloid 2 (TRPV2). We have shown that this receptor is in the cardiomyocyte and report a positive inotropic effect of the drug. Using echocardiography, Langendorff and isolated myocytes, we measured the change in contractility and, using TRPV2(-/-) mice, proved that the effect was mediated by TRPV2 channels in the cardiomyocytes. Analysis of the expression of Ca(2+) handling and β-adrenergic signaling pathway proteins showed that the contractility was not increased through activation of the β-ADR. We propose that the response to probenecid is due to activation of TRPV2 channels secondary to SR release of Ca(2+).

Figure 1

Echocardiographic data. A. Representative B-mode and M-mode from long axis views for baseline and after administration of probenecid 200mg/kg IV. B. Average change (between 5 and 30 minutes) in EF from baseline after IV administration of saline and 200mg/kg probenecid, *P<0.05. C. Time course of the change in EF following IV administration of 200mg/kg probenecid, *P<0.05. D. Dose-dependent changes in EF after injection of various different concentrations of probenecid, measurements were taken every 5 minutes and the change from baseline was average form 5 to 30 minutes. E. Average change in EF following IP administration of 100mg/kg probenecid for wild type (WT), TRPV2^+/− (HET) and TRPV2^−/− (KO), *P<0.05.

Figure 2

Electrocardiographic variables measured after various doses of probenecid were administered, A. RR interval, B. PR interval, C. QRS width and D. Heart rate. There were no statistically significant differences between any of the doses or groups.

Figure 3

In vivo measurement and Langendorff perfused measurements of +dP/dt A. +dP/dt after administration of 30 mg/kg and 5 minutes following 100 mg/kg of probenecid IV, *P<0.05. B. Change in +dP/dt during perfusion of 10⁻⁶ probenecid, 90-210 sec are all significantly different from baseline, *P<0.05.

Figure 4

Quantitative RT-PCR product from mRNA isolated from wild type (WT), TRPV2^+/− (HET) and TRPV2^−/− (KO) mouse hearts.

Figure 5

Western blot analysis of Ca²⁺ handling proteins. Phosphorylated Phospholamban (p-PLN) and phosphorylated Ryanodine receptor 2 (p-RyR2) did not show significant difference between saline in comparison to probenecid.

Figure 6

Effect of probenecid on myocyte contractility. A. Representative contraction traces of ventricular myocytes upon exposure to 10⁻⁷ M probenecid (PROB). B. Dose-response curve of probenecid on myocyte contractility. Data points are averages from 4 mice and fitted to sigmoidal non-linear regression curve. C. Representative contraction traces of ventricular myocytes pretreated with 10⁻⁶ M ruthenium red (RR) and then exposed with 10⁻⁷ M probenecid. D. Average data on myocyte fractional shortening (FS) with ruthenium red pretreatment under control and 10⁻⁷ M probenecid exposure. E. Representative Ca²⁺ transient traces under field stimulation upon exposure 10⁻⁷ M probenecid. F. Average data on Ca²⁺ transient amplitude F/F0 (left) and time constant tau (right) under control and 10⁻⁷ M probenecid exposure. *P<0.001, NS P>0.2.

Figure 7

Effect of probenecid on myocyte cytosolic Ca²⁺. A. Images of confocal microscopic line-scan of cytosolic Ca²⁺ from a myocyte under control and upon exposure to 10⁻⁷ M probenecid for 5 minutes. A heat map is shown to indicate the F/F0 intensity (valued from 0 to 2). B. Average data on Ca²⁺ spark frequency under control and upon exposure to 10⁻⁷ M probenecid for 5 minutes. *P<0.001. C. Time courses of cytosolic Ca²⁺ in myocytes with and without 10⁻⁶ M ruthenium red (RR) pretreatment upon exposure to 10⁻⁷ M probenecid. All the data points are normalized to time 0 and probenecid was applied at 1 minute. D. Average data on myocyte cytosolic Ca²⁺ under control and 10⁻⁷ M probenecid exposure (for 5 minutes) *P<0.001. E. Average data on myocyte cytosolic Ca²⁺ pretreated with 10⁻⁶ M ruthenium red under control and 10⁻⁷ M probenecid exposure (for 5 minutes). NS: P>0.9. F. Patch clamp data from the same myocyte showing no inward Ca²⁺ current with 10⁻⁷ M probenecid treatment (left) and L-type Ca²⁺ current elicited by depolarization voltage step to +10 mV. The myocyte is held at −70 mV. G. Images of confocal microscopic line-scan of cytosolic Ca²⁺ from a myocyte pretreated for 15 minutes with 10⁻⁶ M thapsigargin (TG), before and after treatment with 10⁻⁷ M probenecid for 5 minutes. H. Average data on myocyte cytosolic Ca²⁺ under control and after 10⁻⁷ M probenecid treatment, in myocytes pretreated with 10⁻⁶ M thapsigargin.