Characteristics of the Ca(2+)-dependent inhibition of cyclic AMP accumulation by histamine and thapsigargin in human U373 MG astrocytoma cells

Abstract

1. Histamine, acting on H(1)-receptors, caused a Ca(2+)-dependent inhibition of forskolin- and isoprenaline-induced cyclic AMP accumulation in monolayers of human U373 MG cells (IC(50) 1.3+/-0.3 microM, maximum inhibition 66+/-3%). The inhibition was not reversed by the protein kinase inhibitor K-252A. 2. Thapsigargin also inhibited cyclic AMP accumulation (IC(50) 6.0+/-0.3 nM, maximum inhibition 72+/-1%). In the absence of extracellular Ca(2+) 5 microM thapsigargin caused only a 12+/-2% inhibition of cyclic AMP accumulation. 3. The inhibitory effect of 100 nM thapsigargin on forskolin-stimulated cyclic AMP accumulation was blocked by La(3+) (best-fit maximum inhibition 81+/-4%, IC(50) 125+/-8 nM). In contrast, the inhibitory action of 10 microM histamine was much less sensitive to reversal by 1 microM La(3+) (33+/-5% reversal, compared with 78+/-6% reversal of the inhibition by thapsigargin measured concurrently). However, in the presence of both thapsigargin and histamine the inhibition of cyclic AMP accumulation was reversed by 1 microM La(3+) to the same extent as the inhibition by thapsigargin alone. 4.++Thapsigargin (5 microM)+1 microM La(3+) caused only a 20+/-1% inhibition of histamine-stimulated phosphoinositide hydrolysis. 5. There was no indication from measurement of intracellular Ca(2+) of any persistent La(3+)-insensitive Ca(2+) entry component activated by histamine. 6. The results provide evidence that Ca(2+) entry is required for the inhibition by histamine and thapsigargin of drug-induced cyclic AMP accumulation in U373 MG astrocytoma cells. The differential sensitivity of the inhibitory action of the two agents to block by La(3+) suggests that more than one pathway of Ca(2+) entry is involved.

Figure 1

Isoprenaline-induced cyclic AMP accumulation in U373 MG cells. (A) Concentration-dependence and effect of 10 μM histamine. Points are the weighted means±s.e.mean from six independent experiments (3–6 determinations at each concentration). Incubations were for 1 min. To allow for variations in the magnitude of the stimulation of cyclic AMP accumulation between experiments, response has been expressed as a percentage of the response to 1 μM isoprenaline acting alone (mean stimulation 4.1±0.1 fold of basal), which was measured in every experiment. (B) Time-course of cyclic AMP accumulation induced by 1 μM isoprenaline. Responses are expressed as the percentage of the response to isoprenaline at 4 min, which was measured in every experiment (mean stimulation 7.3±0.2 fold of basal, n=6). Other points are the weighted means±s.e.mean from three determinations.

Figure 2

Ca²⁺-dependence of the inhibition of isoprenaline-stimulated cyclic AMP accumulation by histamine. EGTA (0.2 mM) was present in every incubation, either with or without the addition of 2 mM Ca²⁺. Incubation with 1 μM isoprenaline (Iso)±10 μM histamine (H) was for 4 min. The values are means±s.e.mean from five replicate determinations within a single experiment. The whole experiment was repeated twice further. ^*P<0.001 compared to isoprenaline with Ca²⁺.

Figure 3

Effect of histamine on forskolin- and isoprenaline-stimulated cyclic AMP accumulation as a function of time. (A) Time course of cyclic AMP accumulation stimulated by 10 μM forskolin in the presence and absence of 10 μM histamine. To allow for variations in the response between experiments, the response to forskolin after 4 min incubation has been set equal to 100 (mean stimulation 8.9±0.2 fold of basal, n=6). (B) Variation with time of the inhibition by 10 μM histamine of the responses to 1 μM isoprenaline and 10 μM forskolin. In both panels the points represent the means±approximate s.e.mean of three determinations (five with isoprenaline at 10 min). Basal cyclic AMP accumulation has been subtracted. Where no error bars are apparent the error was within the size of the symbol.

Figure 4

Concentration-dependence of the inhibition by histamine of isoprenaline-stimulated cyclic AMP accumulation. Points are the weighted means±approximate s.e.mean from 2–7 determinations of the ratio of cyclic AMP accumulation in the presence of antagonist to the accumulation in the presence of 1 μM isoprenaline alone (1 min incubation). The curve drawn is the best-fit line to a Hill equation (see Methods).

Figure 5

Effect of protein kinase blockade by K-252A on the inhibition of isoprenaline-stimulated cyclic AMP accumulation by histamine. The bars represent the mean±s.e.mean of quadruplicate determinations within a single experiment in the presence 1 μM isoprenaline (Iso) with or without 1 μM K-252A (K), 10 μM histamine (H) and 1 μM La³⁺ (La), or in the absence of isoprenaline (Basal, B). The whole experiment was repeated twice further with this combination of treatments. ^*Significantly different from isopren-aline+histamine, P<0.001. There was no significant difference between the pairs: basal and basal+K-252A, isoprenaline and isoprenaline+K-252A, isoprenaline+K-252A and isoprenaline+K-252A+La³⁺, isoprenaline+histamine and isoprenaline+histamine+K-252A, isoprenaline+histamine+La³⁺ and isoprenaline+histamine+La³⁺+K-252A.

Figure 6

Inhibition of isoprenaline-stimulated cyclic AMP accumulation by thapsigargin: contribution of Ca²⁺ released from intracellular stores. Incubation with 1 μM isoprenaline (Iso)+5 μM thapsigargin (Tg) in normal Ca²⁺-containing medium or in medium with no added Ca²⁺ and containing 0.2 mM EGTA was for 1 min. The data are the means±s.e.mean of six replicate determinations within a single experiment, which was repeated a further three times. ^*Significantly different from isoprenaline in the absence of Ca²⁺, P<0.01. ^**Significantly different from isoprenaline+Ca²⁺, P<0.001.

Figure 7

Concentration-dependence of the inhibition by thapsigargin of forskolin-stimulated cyclic AMP accumulation in U373 MG cells. Incubation with 10 μM forskolin+thapsigargin was for 4 min. Points are the weighted means±s.e.mean of 2–6 independent determinations. The curve drawn is the best-fit line to a Hill equation (see Methods).

Figure 8

Effect of La³⁺ on the increases in [Ca²⁺]_i in a monolayer of U373 MG cells induced by thapsigargin and histamine. (A) Thapsigargin (100 nM) was added in medium without added Ca²⁺. The cells were superfused with medium containing 1.8 mM Ca²⁺ for the period indicated by the filled horizontal bar. (B) Histamine (10 μM). Ca²⁺ (1.8 mM) and 1 μM La³⁺ were present throughout. The traces in A and B are from single experiments, which were repeated twice further.

Figure 9

Concentration-dependence of the reversal by La³⁺ of the inhibition by 100 nM thapsigargin of cyclic AMP accumulation stimulated by 10 μM forskolin. The cells were incubated with La³⁺ for 10 min before addition of forskolin and further incubation for 4 min. Points are the weighted means from 3–6 independent determinations. The mean stimulation by forskolin was 9.3±0.2 fold of basal (n=9). The curve drawn is the best-fit line to a Hill equation (see Methods), with the foot of the curve set to 28.9%, the weighted mean inhibition in the absence of La³⁺.

Figure 10

Differential effect of La³⁺ on the inhibition by histamine and thapsigargin of cyclic AMP accumulation induced by a 4 min incubation with isoprenaline. Isoprenaline (1 μM) (Iso), 5 μM thapsigargin (Tg), 10 μM histamine (H) and 1 μM La³⁺ were present where indicated. Points are the means±s.e.mean from five replicate determinations within a single experiment. Ca²⁺ (1.8 mM) was present throughout. The whole experiment was repeated once further. ^*Significantly different from isoprenaline+histamine, P<0.01. There was no significant difference between the pairs: isoprenaline and isoprenaline+thapsigargin+La³⁺, isoprenaline+thapsigargin and isoprenaline+histamine, isoprenaline+thapsigargin+La³⁺ and isoprenaline+thapsigargin+histamine+La³⁺.