Activation of β-adrenergic receptors is required for elevated α1A-adrenoreceptors expression and signaling in mesenchymal stromal cells

Abstract

Sympathetic neurons are important components of mesenchymal stem cells (MSCs) niche and noradrenaline regulates biological activities of these cells. Here we examined the mechanisms of regulation of MSCs responsiveness to noradrenaline. Using flow cytometry, we demonstrated that α1A adrenergic receptors isoform was the most abundant in adipose tissue-derived MSCs. Using calcium imaging in single cells, we demonstrated that only 6.9 ± 0.8% of MSCs responded to noradrenaline by intracellular calcium release. Noradrenaline increases MSCs sensitivity to catecholamines in a transitory mode. Within 6 hrs after incubation with noradrenaline the proportion of cells responding by Ca(2+) release to the fresh noradrenaline addition has doubled but declined to the baseline after 24 hrs. Increased sensitivity was due to the elevated quantities of α1A-adrenergic receptors on MSCs. Such elevation depended on the stimulation of β-adrenergic receptors and adenylate cyclase activation. The data for the first time clarify mechanisms of regulation of MSCs sensitivity to noradrenaline.

Figure 1. Expression of functionally active adrenergic receptors in cultured MSCs.

(a) – Representative flow cytometry graphs. Histogram of adrenergic receptors fluorescence showed as red curve, IgG control – as gray curve. (b) – Proportions of cells expressing adrenergic receptors in MSCs of 10 different donors. Each symbol corresponds to one donor. Black line shows median value. (c–e) – Ca²⁺ imaging in MSCs treated with agonists of adrenergic receptors. (c) – Representative recordings of intracellular Ca²⁺ in individual cell, treated with noradrenaline in series (see also Supplemental Movie 2). (d,e) – Representative recordings of intracellular Ca²⁺ in individual cell, treated with noradrenaline in series in the presence of antagonists of α1- (10 μM of prazosin) and α2- (100 μM of atipamezole) adrenergic receptors. Short lines above fluorescence traces show applications of indicated compounds.

Figure 2. Noradrenaline increased the proportion of MSCs responsive to the hormone.

(a) – Proportion of MSCs capable to respond to noradrenaline. NA – cells stimulated by noradrenaline for 1 hr, washed three times and incubated in full growth medium for 5 hrs followed by loading with Fluo-8 and imaging. Control - fresh growth medium was added instead of noradrenaline. Mean ± s.e.m., n = 28, ***p < 0.001 calculated with Mann-Whitney Rank Sum Test. (b,c) – Superimposed dose dependences of noradrenaline responses recorded from 8 control cells (b) and 13 noradrenaline treated cells (c). Responses of a given cell were normalized to a response induced by 1 μM noradrenaline; each symbol corresponds to an individual cell. Median values are indicated by vertical gray lines. (d) – Proportion of the cells responded to α1-agonist phenylephrine (100 μM) after noradrenaline treatment; n = 12, *p < 0.05 calculated with Mann-Whitney Rank Sum Test. (e) –Proportion of the cells responded to α2-agonist clonidine (100 μM) after noradrenaline treatment. Mean ± s.e.m., n = 10.

Figure 3. Noradrenaline treatment increased an amount of α1A-adrenergic receptors.

(a) – mRNA of adrenergic receptors 6 hrs after noradrenaline treatment (real-time PCR). Mean ± s.e.m., n = 5, *p < 0.05 calculated with One Way ANOVA. (b) – Proportions of MSC expressing particular adrenergic receptors after noradrenaline treatment (NA) vs. untreated cells (Control). Mean ± s.e.m., n = 8. (c) – Representative flow cytometry graphs (donors 3, 4 and 8) of α1A-adrenergic receptors in MSC treated with noradrenaline (blue curves) versus cells pretreated with growth medium only (red curves). IgG controls showed as gray curves. (d) – The mean fold-change of the fluorescence intensity in MSCs treated with noradrenaline as compared to vehicle treated cells. Mean ± s.e.m., n = 8, *p < 0.05 calculated with Kruskal-Wallis One Way ANOVA on Ranks. (e) – Representative cropped Western Blot membranes of adrenergic receptors isoforms content in MSCs treated with noradrenaline or vehicle. Full-length blots are presented in Supplementary Fig. S6. (f) – The mean fold-change in the Western Blot adrenergic receptors bands intensity of noradrenaline pretreated cells as compared to vehicle treated cells. Mean ± s.e.m., n = 10, *p < 0.05 calculated with Kruskal-Wallis One Way ANOVA on Ranks.

Figure 4. β-adrenergic receptors and adenylate cyclase regulated the number of MSC responding to noradrenaline.

(a) – Proportion of MSCs responding to noradrenaline 6 hrs after pretreatment with adrenergic receptors agonists (1 μM of noradrenaline (NA), 100 μM of α1-agonist phenylephrine (Phe), 100 μM of α2-agonist clonidine (Clon), 1 μM of β-agonist dobutamine (Dob) or Vehicle (Cont)) instead of noradrenaline. Mean ± s.e.m., n = 14–21, *p < 0.05 calculated with Kruskal-Wallis One Way ANOVA on Ranks. (b) – Proportion of MSCs responding to noradrenaline 6 hrs after pretreatment with noradrenaline with adrenergic receptors antagonists (10 μM of α1-antagonist prazosin (Praz), 100 μM of α2-antagonist atipamezole (Atip), 10 μM of β-antagonist alprenolol (Alpr) or Vehicle (Cont)). Mean ± s.e.m., n = 6–12, *p < 0.05 calculated with Kruskal-Wallis One Way ANOVA on Ranks. (c) – Proportion of MSCs responding to noradrenaline 6 hrs after pretreatment with adenylate cyclase activator forscolin (1 μM) or by noradrenaline with 1 μM of adenylate cyclase inhibitor SQ22536 (SQ). Mean ± s.e.m., n = 3–6, *p < 0.05 calculated with Kruskal-Wallis One Way ANOVA on Ranks. (d) – α1A-adrenergic receptors in MSCs treated with noradrenaline (NA) or noradrenaline together with SQ22536 (SQ). Full-length blots are presented in Supplementary Fig. S6. The mean fold-change in the Western Blot bands intensity of NA pretreated cells as compared to vehicle treated cells. Mean ± s.e.m., n = 5, *p < 0.05 calculated with Mann-Whitney Rank Sum Test.