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ISSN: 2161-0479
Journal of Autacoids and Hormones

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Physiologic Importance Of In Vitro Studies With Autacoids

Mary Taub*
Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, USA
Corresponding Author : Mary L. Taub
Biochemistry Dept., 140 Farber Hall, University at Buffalo
3435 Main Street, Buffalo, New York 14214, USA
E-mail: [email protected]
Received August 23, 2012; Accepted August 24, 2012; Published August 26, 2012
Citation: Taub M (2012) Physiologic Importance of In Vitro Studies with Autacoids. J Autacoids 1:e119. doi: 10.4172/2161-0479.1000e119
Copyright: © 2012 Taub M. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Autacoids are effector molecules that act on cells close to the locale of their synthesis [1]. In some cases autacoids also have the potential to enter the circulation, so as to affect cells in other tissues [1]. Early studies were concerned with eicosanoids, lipid acids derived from aracidonic acid via either Cyclooxygenase (COX) or Lipoxygenase (LOX) [2]. Prostaglandins (PGs), products of COX, initially isolated from seminal fluid, were subsequently found in numerous tissues. Many different classes of autacoids are presently being studied, including angiotensin, norepinephrine, dopamine, nitric oxide, endothelins, Insulin-Like- Growth Factor (IGF), Epidermal Growth Factor (EGF) and Fibroblast Growth factor (FGF), to name a few. Of particular interest to this report, is the use of cultured animal cells to study the effects of autacoids.
A question that arises in these regards is whether observations of the effects of autacoids in vitro are physiologically relevant. In initial cell culture studies, investigators were concerned whether differentiated cells actually dedifferentiated when cultured, and became fibroblasts [3]. However, the problem of fibroblast overgrowth of primary cultures was subsequently found to be the consequence of the use of serum in the culture medium which gave a select advantage to fibroblasts and in some cases was actually toxic to differentiated cells. When using defined medium lacking serum, differentiated cells may be grown selectively in primary cultures in the absence of fibroblasts. Under these conditions, physiologically relevant effects of autacoids may be observed [4]. Similar responses may be difficult to discern in vivo, especially when the physiologic environment is rapidly changing.
Presently, a number of in vitro cell culture systems are available, which are highly differentiated, and closely resemble normal cells present in their tissue of origin [5]. A number of growth studies have been conducted with these differentiated cell culture systems in hormonally defined media [3,6]. In the absence of serum, the growth requirements of differentiated cells have been found to differ, in a tissue-specific manner. A number of autacoids have been observed to stimulate growth in defined medium, also in a tissue specific manner, suggestive that the cells may have similar requirements in vivo. One example of such an autacoid is, putrescine, a growth factor for B104 rat neuroblastoma cells, as well as primary neurons [7,8]. Consistent with an in vivo requirement, putrescine is detectable in brain [9] and has been reported to stimulate brain neurogenesis, as well as neurite outgrowth [10]. Hormones may similarly stimulate the growth of cultured animal cells, as exemplified in the case of Follicle Stimulating Hormone (FSH), which is a growth factor for rat Sertoli cells in culture [4]. This makes physiologic sense, because after FSH is produced by the gonadotrophic cells in the anterior pituitary [11], FSH acts on Sertoli cells within the seminiferous tubules of the testis, so as to promote spermatogenesis [11]. Increases in the number of Sertoli cells are observed following the production of FSH, in addition to increases in the level of the FSH receptor.
Another class of autacoids, PGs, stimulate the growth of a number of cell lines in vitro [4,12]. For example, the growth of the dog kidney tubule epithelial cell line MDCK is stimulated by either PGE1 or PGE2 [13]. Consistent with the physiologic significance of the growth stimulatory effects of PGE1 and PGE2 is the observation that the growth of primary baby mouse kidney cells (taken directly from the animal) is stimulated by the same PGs [14]. The growth of epithelial cells derived from a number of other tissues is similarly stimulated by PGE2, including mammary epithelial cells [15], corneal epithelial cells [16], and intestinal epithelial cells [17]. Indeed, increased growth of colon cancer cells has been attributed to increased production of PGE2 by the tumor [18].
PGs have a number of functional affects. In the kidney, PGs affect vascular tone, as well as epithelial transporters [19]. However, the functional effects of PGs observed in vitro, are often difficult to validate in vivo, due to complexity of organs such as the kidney. The kidney is composed of numerous nephrons, each of which has distinct segments. Due to the convoluted structure of the nephrons, they cannot be purified simply by cutting out kidney slices. Instead, individual nephron segments are isolated. Using such individual nephron segments, the effects of PGE2, the major renal PG, have been examined by means of microperfusion studies [20]. The results of these studies indicate that PGE2 does indeed affect ion transport, although the effects differ significantly between nephron segments [20]. The differences can be explained by the presence of different transporters, as well as different sets of receptors for PGE2 (EP receptors) in each nephron segment.
A classic example of such PG affects is the inhibitory affect of PGE2 on Arginine Vasopressin (AVP) induced increase in water permeability in Collecting Duct (CD) Principle Cells [21]. The inhibitory effect of PGE2 on water reabsorption in the CD has been proposed to be mediated by Gi-coupled EP3 receptors (localized in this nephron segment). Indeed, the EP3 agonist sulprostone antagonizes the effect of AVP on water reabsorption by the kidney [21]. This is apparently a major effect, as Non steroidal Anti-inflammatory Drugs (NSAIDS) cause water retention [19]. Nevertheless, mice with a targeted disruption in EP3 only subtly alter the water retention caused by NSAIDs [19]. Compensatory effects caused by other EP receptors were postulated. Indeed, the Gq-coupled EP1 has also been proposed to play a role in the collecting duct. The EP1 agonist 17-phenyl-trinor-PGE2 was observed to inhibit water reabsorption, and this effect was prevented by SC- 19920, an EP1 antagonist [22]. To date the relative roles of EP1 and EP3 in the collecting duct are unresolved. In vitro studies with isolated cells from the CD would be an ideal way to clarify these issues.
Although, Gs-coupled EP2 and EP4 receptors are also present in the kidney [19], in vivo studies have not elucidated their roles. However, in vitro studies with the MDCK cell line, a distal tubule model, and primary rabbit renal proximal tubule (RPT) cells have indicated that EP2 and EP4 are involved in Na, K-ATPase regulation [23]. All 4 EP receptors (EP1, EP2, EP3 and EP4) are present both in MDCK cells and primary rabbit RPT cells [24,25]. Evidence for chronic stimulatory effects of PGE1 and PGE2 on Na, K-ATPase has been obtained in both renal cell types [23,25]. The increased rate of Na,KATPase biosynthesis observed in MDCK cells treated with PGE1 have been explained by transcriptional regulation of the Na,K-ATPase β subunit gene (atp1b1) via Prostaglandin Response Elements [23,26,27]. Evidence for the involvement of both Gq-coupled EP1 and Gs-coupled EP2 in the transcriptional regulation of atp1b1was obtained initially [23]. Subsequently, similar transcriptional studies were conducted in the primary rabbit RPT cells, and additional evidence was obtained for EP4 involvement [25].
Although PGE1 and PGE2 are also growth stimulatory factors for these renal cells, our studies with genetic variants of MDCK indicate that distinct cAMP mediated mechanisms are involved in mediating the stimulatory effects of these PGs on growth [28]. Studies concerning growth and transport are still lacking in intact animals. Nevertheless, the results with Na, K-ATPase are consistent with previous studies of Na,K-ATPase in isolated proximal convoluted and distal tubules [29].
In the cell culture studies described above, similar results were obtained with primary cultures obtained directly from the animal and an established cell line [25,26]. When experiments are conducted with established cell lines in vitro, in the absence of other modes of confirmation, caution must be taken with regards to the conclusions. Animal cell lines may have changed while being established in culture. In addition, cell function may be affected by immortalizing oncogenes. Indeed, following immortalization with SV40 early region genes, rabbit RPT cell lines had considerably lower rates of sugar uptake than primary RPT cell cultures, and did not grow in glucose free medium (unlike primary cultures), indicating the loss of gluconeogenic capacity [30].
The cell culture environment is another concern when evaluating the results obtained in vitro. Imperfections in the in vitro environment (which do not effectively simulate the in vivo situation) may result in cellular adaptations, which are necessary for cell proliferation and survival. In this case, concern must be raised about the physiologic relevance of effectors and signaling pathways in studies with such immortalized cells.
To summarize, in vitro studies concerning the effects of autacoids on differentiated animal cells have potential to make significant contributions to our understanding their signal transduction pathways in vivo. The use of hormonally defined culture medium makes it possible to study the effects of autacoids at physiologically relevant concentrations, and to maintain cultures in their differentiated state. Conclusions obtained with immortalized cell lines should be assessed using other model systems including primary cultures, as signaling pathways and hormonal responses may be altered as a consequence of cell immortalization. With these constraints in mind, in vitro studies with differentiated cells in defined culture conditions have a great potential to further our understanding of the in vivo situation, and provide an intelligent rationale for therapeutics.
 
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