Alpha 1-adrenoceptor effects mediated by protein kinase C alpha in human cultured prostatic stromal cells

Abstract

1 We have investigated the effects of alpha(1)-adrenoceptor stimulation upon contractility, Ca(2+) influx, inositol phosphate production, and protein kinase C (PKC) translocation in human cultured prostatic stromal cells (HCPSC). 2 The alpha(1)-adrenoceptor selective agonist phenylephrine elicited contractile responses of HCPSC, i.e. a maximal cell shortening of 45+/-6% of initial cell length, with an EC(50) of 1.6+/-0.1 microM. The alpha(1)-adrenoceptor selective antagonists prazosin (1 microM) and terazosin (1 microM) both blocked contractions to phenylephrine (10 microM). The L-type calcium channel blocker nifedipine (10 microM), and the PKC inhibitors Gö 6976 (1 microM) and bisindolylmaleimide (1 microM) also inhibited phenylephrine-induced contractions. 3 Phenylephrine caused a concentration dependent increase in inositol phosphate production (EC(50) 119+/-67 nM). This response was blocked by terazosin (1 microM). 4 Phenylephrine caused the translocation of the PKC alpha isoform, but not the beta, delta, gamma, epsilon or lambda isoforms, from the cytosolic to the particulate fraction of HCPSC, with an EC(50) of 5.7+/-0.5 microM. 5 In FURA-2AM (5 microM) loaded cells, phenylephrine elicited concentration dependent increases in [Ca(2+)](i), with an EC(50) of 3.9+/-0.4 microM. The response to phenylephrine (10 microM) was blocked by prazosin (1 microM), bisindolymaleimide (1 microM), and nifedipine (10 microM). 6 In conclusion, this study has shown that HCPSC express functional alpha(1)-adrenoceptors, and that the intracellular pathways responsible for contractility may be largely dependent upon protein kinase C activation and subsequent opening of L-type calcium channels.

Figure 1

The effects of phenylephrine upon human cultured prostatic stromal cell contractility. Results are expressed as percentage reduction in initial cell length. (a) Shows a typical response to phenylephrine 10 μM, scale bar represents 200 μm and calipers indicate where cell length measurements were taken from, (i) immediately before agonist addition, and (ii) 30 min after agonist addition. (b) Shows the concentration–response curve to phenylephrine, (n=8). ^*Significant when compared to vehicle control, P<0.05; one-way ANOVA.

Figure 2

The effects of the α₁-adrenoceptor selective antagonists, prazosin and terazosin upon phenylephrine-stimulated contractility in human cultured prostatic stromal cells. Cells were incubated with either prazosin (1 μM), terazosin (1 μM) or vehicle control prior to the addition of phenylephrine (PE, 10 μM). In all cases n=6. ^†Significant when compared to vehicle control, P<0.05; Dunnett's test. ^*Significant when compared to phenylephrine (10 μM), P<0.05; Bonferroni's test.

Figure 3

The effects of the protein kinase C inhibitors Gö 6976 and bisindolylmaleimide, and the L-type calcium channel blocker nifedipine upon phenylephrine-stimulated contractility in human cultrued prostatic stromal cells. Cells were incubated with either Gö 6976 (1 μM), bisindolymaleimide (1 μM), nifedipine (NIF, 10 μM) or vehicle control prior to the addition of phenylephrine (10 μM). In all cases n=6. ^†Significant when compared to vehicle control P<0.05; Dunnett's test. ^*Significant when compared to PE (10 μM), P<0.05; Bonferroni's test.

Figure 4

The effects of phenylephrine and terazosin upon [³H]-inositol phosphate accumulation in human cultured prostatic stromal cells. Cells were incubated with [³H]-myo-inositol and either terazosin 1 μM or vehicle control prior to the addition of phenylephrine (PE). In all cases n=5. ^*Significant when compared to vehicle control P<0.05; one-way ANOVA.

Figure 5

Shows the effects of phenylephrine upon PKC translocation in human cultured prostatic stromal cells. (a) Shows a typical Western blot, demonstrating translocation of protein kinase C α from cytosolic to particulate fractions in response to vehicle control or phenylephrine (100 μM). (b) Shows mean translocation ratio of protein kinase C α in response to PE (100 nM–100 μM). ^*Significantly different when compared to vehicle control, P<0.05; one-way ANOVA, n=4).

Figure 6

The effects of phenylephrine upon intracellular Ca²⁺ ([Ca²⁺]_i) in human cultured prostatic stromal cells. Cells were incubated with the FURA-2AM (5 μM) prior to the addition of phenylephrine. (a) Shows mean changes in [Ca²⁺]_i over 30 min, in response to phenylephrine. (b) Shows concentration–response curve showing changes in [Ca²⁺]_i 25 min after the addition of phenylephrine. In all cases n=6. ^*Significantly different when compared to vehicle control P<0.05; one-way ANOVA.

Figure 7

The effects of nifedipine, bisindolymaleimide and prazosin upon phenylephrine-stimulated increases in intracellular Ca²⁺ ([Ca²⁺]_i) in human cultured prostatic stromal cells. Cells were incubated with the FURA-2AM (5 μM) prior to the addition of phenylephrine. Antagonists and blockers were added 45–60 min prior to the addition of phenylephrine. (a), (b) and (c) Show mean changes in [Ca²⁺]_i over 30 min, in response to phenylephrine (10 μM), in the presence and absence of prazosin (1 μM), bisindolymaleimide (1 μM) and nifedipine (10 μM) respectively. ^*Significant when compared to phenylephrine (10 μM). P<0.05, Dunnett's test (n=6).